WO2024054769A1 - Méthodes de génération d'un produit de cellules monocytes/macrophages à partir de cellules souches et progénitrices hématopoïétiques cd34+ issues du sang de cordon multipliées - Google Patents

Méthodes de génération d'un produit de cellules monocytes/macrophages à partir de cellules souches et progénitrices hématopoïétiques cd34+ issues du sang de cordon multipliées Download PDF

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WO2024054769A1
WO2024054769A1 PCT/US2023/073151 US2023073151W WO2024054769A1 WO 2024054769 A1 WO2024054769 A1 WO 2024054769A1 US 2023073151 W US2023073151 W US 2023073151W WO 2024054769 A1 WO2024054769 A1 WO 2024054769A1
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cells
cell
receptor
macrophage
monocyte
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Katherine BREMPELIS
Colleen Delaney
Erika VON EUW
Shannon KREUSER
Carrie STOLTZMAN
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Deverra Therapeutics Inc.
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4614—Monocytes; Macrophages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/15—Cells of the myeloid line, e.g. granulocytes, basophils, eosinophils, neutrophils, leucocytes, monocytes, macrophages or mast cells; Myeloid precursor cells; Antigen-presenting cells, e.g. dendritic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4622—Antigen presenting cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464411—Immunoglobulin superfamily
    • A61K39/464412—CD19 or B4
    • C—CHEMISTRY; METALLURGY
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0645—Macrophages, e.g. Kuepfer cells in the liver; Monocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/125—Stem cell factor [SCF], c-kit ligand [KL]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/145—Thrombopoietin [TPO]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/22—Colony stimulating factors (G-CSF, GM-CSF)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2303—Interleukin-3 (IL-3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/26—Flt-3 ligand (CD135L, flk-2 ligand)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/11—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from blood or immune system cells

Definitions

  • the Sequence Listing XML associated with this application is provided in XML format and is hereby incorporated by reference into the specification.
  • the name of the XML file containing the sequence listing is 1765-P13WO_Seq_List_20230829.xml.
  • the XML file is 90,003 bytes; was created on August 29, 2023; and is being submitted electronically via Patent Center with the filing of the specification.
  • the present disclosure relates to methods for the ex vivo expansion of hematopoietic stem cells and/or hematopoietic progenitor cells (HSPCs) and the differentiation of the expanded HSPCs ex vivo to form a cell product comprising functional monocytes and macrophages.
  • the HSPCs can be CD34+ cells which are derived from one or more human subjects.
  • the CD34+ cells can be derived from human cord blood and/or placental blood.
  • the monocyte/macrophage cell product generated by the disclosed ex vivo methods from pooled unmatched, matched, or partially mismatched, cord blood and/or placental blood CD34+ stem and progenitor cells comprises ⁇ 70 % functional CDl lb+ HLA-DR+ monocytes and macrophages and ⁇ 30 % other myeloid cells, including granulocytes and dendritic cells.
  • These monocytes and/or macrophage cell products are also comprised of about 70 % CD14 CD16 double positive cells.
  • the monocytes and macrophages that comprise the cell product demonstrate functional phagocytic capacity in vitro.
  • the cell product produced by the disclosed methods can be used as a therapeutic agent against tumor cells, including both hematologic and solid tumors. This product can also be used as an antimicrobial therapeutic and as a therapeutic in repair of injured tissue.
  • the produced cell product can also be used for autoimmune indications. Its specificity and/or potency can also be improved through, for example, genetically engineering the cells to express, for example, a receptor (such as a chimeric antigen receptor (CAR), a T cell receptor (TCR), a chimeric receptor, or other receptor), an immune cell engager, an antibody, a nanobody, a transcription factor, a cytokine, and/or a chemokine.
  • the function of the cell product can be improved by pre-loading the cells with an oncolytic virus or other immunomodulatory molecule for delivery to a solid tumor.
  • Macrophages constitute a heterogeneous cell population representing innate immunity. Discovered at the end of 19th century by Ilya Mechnikov, macrophages have been identified in all tissues. Their chief competences are phagocytic activity and antigen presentation. Macrophages continuously monitor their microenvironments for the presence of pathogens, unfit cells, debris, and toxic metabolites, and release a variety of active substances including growth factors and cytokines. Human macrophages express several markers including CD14, CD16, CD68, CD163, CD1 lb, CD86, and CD206. According to the traditional concept, macrophages are classified into pro-inflammatory (Ml), non-activated (MO) or anti-inflammatory (M2) subsets that play distinct roles in the initiation and resolution of inflammation.
  • Ml pro-inflammatory
  • MO non-activated
  • M2 anti-inflammatory
  • Macrophages are cells involved in fundamental biological processes, including inflammation development and homeostasis support. They mediate host protection by engulfing and eliminating pathogens, by secreting a wide range of proinflammatory mediators that attract and activate immune cells at the site of infection, and by processing and presenting antigens to T lymphocytes, which propagates an adaptive immune response in the tissues.
  • Macrophages are also able to limit inflammation and mediate tissue repair and wound healing, largely by secreting anti-inflammatory and tissue remodeling factors and by phagocytizing apoptotic and necrotic cells.
  • the foundation for the various and often opposite activities is formed by macrophages’ capacity to sense the microenvironment and finetune their transcriptomic and functional programs according to homeostatic requirements. Dysregulation of these processes underlies many diseases. An exacerbated inflammatory response and/or impaired phagocytic/clearance activities of macrophages have been implicated in the pathogenesis of autoimmune, chronic inflammatory, cardiovascular, metabolic, neurodegenerative, infectious, and several hereditary diseases.
  • Adoptive cell therapy represents a promising approach to support standard cancer treatment strategies, including surgery, radiation, and chemotherapy. Efforts to strengthen the patient’s immune system through administration of cellular immune therapeutics have resulted in significant improvements for cancer patients.
  • the chimeric antigen receptor (CAR) T cell therapy has recently revolutionized the fight against hematologic malignancies.
  • CARs are artificial receptors that consist of an extracellular antigen-binding unit, e.g., derived from a single chain antibody fragment (scFv), a hinge region, a transmembrane sequence, and an intracellular signaling domain responsible for specific cell activation.
  • CAR principles were also applied to other immune cell types, such as natural killer (NK) cells, which showed encouraging results in first clinical trials in patients with CD19+ lymphoid tumors.
  • NK natural killer
  • Macrophages can infiltrate solid tumors, actively attack cancer cells, and simultaneously orchestrate important immune responses and activate bystander cells to enhance the anti-tumor response. Furthermore, they are capable of cytokine secretion and exhibit a high loading capacity, which can be exploited for use of these cells as cargo vehicles. Macrophages were used as adoptive cell therapies against solid tumors and autologous transfer of blood-derived monocytes was shown to be safe.
  • macrophage cells ex vivo, such as generation from induced pluripotent stem cells, from isolated peripheral blood mononuclear cells, from peripheral blood stem or progenitor cells, or from umbilical cord blood stem or progenitor cells.
  • Macrophages can be used in vivo as a form of cell therapy, either unmodified or genetically modified to express a receptor (such as a CAR, a TCR, a chimeric receptor, or other receptor), an immune cell engager, an antibody, a nanobody, a transcription factor, a cytokine, and/or a chemokine to treat various cancer types, or infectious, inflammatory, and/or autoimmune diseases.
  • a receptor such as a CAR, a TCR, a chimeric receptor, or other receptor
  • an immune cell engager an antibody, a nanobody, a transcription factor, a cytokine, and/or a chemokine to treat various cancer types, or infectious, inflammatory, and/or autoimmune diseases.
  • the present disclosure provides methods for preparing compositions and preparation comprising monocytes and/or macrophage for use in immunotherapy.
  • a method of preparing a monocyte and/or macrophage preparation for use in immunotherapy comprising: selecting a plurality of umbilical cord blood or placental blood cells; preparing enriched CD34+ hematopoietic stem and progenitor cells (HSPCs) that are depleted of red blood cells and T cells; culturing the CD34+ enriched HSPCs in an expansion culture medium comprising interleukin-3 (IL-3), interleukin-6 (IL-6), thrombopoietin (TPO), Flt-3 ligand (Flt-3L), and stem cell factor (SCF) on a solid phase for a sufficient time to produce expanded HSPCs, wherein the expanded HSPCs do not substantially differentiate into CD1 lb+ HLA-DR+ cells during the expansion; and culturing the expanded HSPCs in an expansion and mon
  • IL-3
  • the umbilical cord blood or placental blood cells used in the method can be from a non-HLA matched, matched, or partially mismatched donor or can be from umbilical cord blood or placental blood cells pooled from non-HLA matched, matched, or partially mismatched donors.
  • the expansion culture step of the method can further comprise a Notch ligand and fibronectin and the expansion culture can be carried out for about 3 to about 21 days.
  • the expansion and monocyte differentiation culture phases are carried out for about 7 to about 14 days.
  • the macrophage differentiation phase is carried out for about 6 to 8 days.
  • the non-animal serum replacement is human AB serum, human serum albumin, or human platelet lysate.
  • the HSPCs are not derived from somatic cells, embryonic stem cells, peripheral blood mononuclear cells or induced pluripotent stem cells.
  • the Notch ligand is DXI or an antibody specific for Notch.
  • cells of the monocyte and/or macrophage composition and/or preparation are genetically modified.
  • the genetic modification is during the expansion phase or subsequent to differentiation of the HSPCs, monocytes and/or macrophage.
  • the monocytes and/or macrophages of the composition and/or preparation are genetically modified to affect a gene knockdown, knockout, or knock-in.
  • the gene targeted for knockdown or knockout is B2M, CIITA, a cytokine, a cytokine receptor, a chemokine, a chemokine receptor, a transcription factor, a growth factor receptor, an immunomodulatory molecule, an immune stimulatory molecule, an immune costimulatory molecule, and/or an immune costimulatory ligand.
  • the gene targeted is HLA-E, HLA-E and B2M, a cytokine, a cytokine receptor, a chemokine, a chemokine receptor, a transcription factor, a growth factor receptor, an immunoregulatory molecule, an immune stimulatory molecule, an immune costimulatory molecule, and/or an immune costimulatory ligand.
  • the cells of the monocyte and/or macrophage composition and/or preparation are genetically modified to express an RNA, an enzyme, a receptor, a chimeric receptor, an immune cell engager, an antibody, a nanobody, a transcription factor, a cytokine and/or a chemokine.
  • the RNA is a shRNA, a siRNA or a gRNA.
  • the receptor is a CAR or a TCR.
  • the receptor, chimeric receptor, immune cell engager, antibody, or nanobody specifically bind to a viral antigen, a bacterial antigen, a tumor- specific, a tumor-associated, or stroma antigen.
  • the viral antigen can be present in a Cytomegalovirus (CMV), an Epstein Barr Virus (EBV), a Human Immunodeficiency Virus (HIV), a Herpes simplex virus (HSV), a Hepatitis virus, a Zika virus, an influenza virus, or a coronavirus.
  • the Herpes virus can be HSV 1 or HSV 2, the Hepatitis virus is Hepatitis A, B, or C, and the coronavirus is SARS-CoV or SARS-CoV-2.
  • the tumor- specific antigen, tumor-associated antigen, or stroma antigen is AFP, ALPP, ALPP2, ANTXR1, alpha- V beta-3 integrin, alpha-V beta-6 integrin, AXL, BCMA, B7-H3 (CD276), B7-H4 (VTCN1), carbonic anhydrase IX (CA1X), carcinoembryonic antigen (CEA), CD5, CD8, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD44V6, CD47, CD49c, CD49e, CD49f, CD56, CD61, CD66c, CD70, CD72, CD73, CD74, CD80, CD86, CD104,CD123, CD126, CD133, CD138, CD142, CD147, CD318, an antigen of a cytomegalovirus (CMV) infected cell (e.g., a cell surface antigen of a cytomegal
  • the tumor- specific or tumor- associated antigen is EGFR or any variant thereof, a NKG2D ligand, HER2, B7-H3, PSMA, PSCA, MUC1 or a variant thereof, mesothelin, or CEA.
  • the CAR comprises an extracellular antigen binding domain, a hinge region, a transmembrane domain, and one or more intracellular signaling domains.
  • the intracellular signaling domain can comprise one or more cytoplasmic or intracellular signaling domains of CD3zeta (CD3Q, CD3delta, CD3epsilon, CD3gamma, CD4, CD8A, CD8B, CD2, CD7, LIGHT, CD27, CD28, 4-1BB (CD137), CD226 (DNAM1), B24 (CD244), ICOS (CD278), CTLA-4, GITR, OX40 (CD134), LAT, PD-1, TIM3, TIGIT, PD-L1, PD-L2, OX40L, 4-1BBL, ICOSLG, CD30L, CD30, CD36, CD68, CD40, CD70, CD80, CD83, CD86, CD163, CD204 (MSR1), CD206 (MRC1), CD209 (DC).
  • TNFRSF1A TNFRSF1B
  • a chemokine receptor comprising CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CCR11, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, XCR1, CX3CR1, or any combination thereof
  • a transmembrane domain comprising a transmembrane domain of an intracellular signaling domain, and optionally comprises CD8, CD28, CD3zeta, CD4, 4-1BB, OX40, ICOS, or NKG2D
  • a spacer region comprising a hinge region of IgGi, the CH2CH3 region of an immunoglobulin, a portion of CD3, a portion of CD28, or a portion of CD 8.
  • the immune cell engager specifically binds to one or more T cell surface proteins, one or more NK cell surface proteins, or one or more monocyte and/or macrophage surface protein.
  • the T cell surface protein preferably is CD3, CD28, and/or 4- IBB .
  • the NK cell surface protein is CD56, CD16, and/or NKG2D.
  • the monocyte and/or macrophage surface protein is CD64, CD40, CD80, and/or CD86.
  • the transcription factor is a C/EBP transcription factor, a NF-KB transcription factor, a STAT transcription factor, a KLF transcription factor, a PPAR transcription factor, an AP-1 transcription factor, a NF AT transcription factor, a GAT A transcription factor, a CREB transcription factor, or an IRF transcription factor.
  • the cytokine or chemokine is an Interleukin, Interferon a, Interferon p, Interferon y, Interferon X, TGF p, TNF a, TNF p, GM-CSF, M-CSF, CCE1, CCE2, CCE3, CCE4, CCE5, CCE7, CCE8, CCL11, CCE13, CCE14, CCE15, CCE16, CCE17, CCE18, CCE19, CCE20, CCE21, CCE22, CCE23, CCE24, CCE25, CCE26, CCE27, CCE28, XCE1, XCE2, CX3CE1, CXCE1, CXCE2, CXEC3, CXCE4, CXCE5, CXCE6, CXCE7, CXCE8, CXCE9, CXCE10, CXCL11, CXCE12, CXCE13, CXCE14, CXCE16, or CXCE17.
  • the interleukins of these embodiments of the method can be IE-1 a, IE-ip, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-17, IL-18, IL-21, or IL-22.
  • the monocyte and/or macrophage composition and/or preparation further comprises a cryoprotective agent.
  • the monocyte and/or macrophage composition and/or preparation is formulated to form a monocyte and/or macrophage formulation for infusion into a subject.
  • the present disclosure also provides a composition comprising the monocyte and/or macrophage composition and/or preparation produced by any of the methods above for use in immunotherapy.
  • Immunotherapy as used herein comprises use of the described compositions and/or preparations as a therapeutic agent against tumor cells, use as an antimicrobial agent, use for autoimmune indications, or use as a therapeutic in repair of injured tissue.
  • the composition comprises the monocyte and/or macrophage composition and/or preparation produced by any of the methods described herein for use in delivery of small molecules, plasmid DNA, oncolytic virus, or other therapeutics.
  • compositions comprising the monocyte and/or macrophage composition and/or preparation produced by any of the methods described herein for use in combination with other therapeutic compositions comprising unmodified or genetically modified T cells or NK cell therapies, antibody or nanobody therapeutics, immune cell engagers, cytokines, chemokines, and/or oncolytic virus.
  • FIGURE 1 A and IB depict the ex vivo expansion of pooled cord blood CD34+ cells.
  • FIGURE 1A shows approximately 78,000 cells were generated from each CD34+ cell seeded into culture on day 0.
  • FIGURES 2A and 2B depict the ex vivo differentiation and expansion of monocytes and macrophages from pooled cord blood CD34+ cells.
  • FIGURE 2A shows the significant differentiation of cells toward becoming HLA-DR+ CDl lb+ occurs during Phase 2 and Phase 3 of culture.
  • FIGURES 3 A through 3C show the HLA-DR and CD 11b co-expression increases significantly during monocyte differentiation (end of Phase 2; FIGURE 3B) and is maintained during macrophage differentiation (end of Phase 3, FIGURE 3C) in the presence of either fetal bovine serum (FBS) or human platelet lysate (HPL).
  • FBS fetal bovine serum
  • HPL human platelet lysate
  • FIGURES 4A through 4D demonstrate that HLA-DR+ CDl lb + macrophages generated ex vivo express markers associated with Ml (FIGURES 4 A and 4B) or M2 (FIGURES 4C and 4D) polarization when differentiated in either GM-CSF or M-CSF, respectively, during Phase 3 of culture.
  • FIGURE 5 demonstrates that ex vivo generated macrophages effectively phagocytose pHrodoTM Staphylococcus aureus BioParticlesTM in an in vitro phagocytosis assay. Bioparticles become fluorescent within the acidic environment of the macrophage cytoplasmic phagosomes.
  • FIGURE 6 depicts macrophage generated ex vivo using either GM-CSF or M-CSF during Phase 3 of culture can be cryopreserved with high levels of post-thaw cell recovery and viability.
  • FIGURE 7 depicts an in vitro phagocytosis assay using fluorescent pHrodoTM S. aureus BioParticlesTM. Monocytes harvested and cryopreserved at the end of Phase 2 of culture or macrophage harvested and cryopreserved at the end of Phase 3 of culture each demonstrate phagocytosis following cryopreservation and thaw.
  • FIGURE 8 shows the cell surface expression of CD 19 CAR on monocytes and macrophages generated by ex vivo expansion and differentiation of CD34+ HSPCs transduced with a CD 19 CAR viral vector.
  • FIGURES 9A though 9C demonstrate that engineering monocytes and macrophages to express a CD 19 CAR does not reduce their total fold expansion (Figure 9 A), cell viability (Figure 9B), or phagocytic capacity (Figure 9C) compared with control un-transduced monocytes and macrophages.
  • the present disclosure provides herein a culture method for the ex vivo expansion of hematopoietic stem and/or hematopoietic progenitor cells (HSPCs) followed by the ex vivo differentiation of the expanded HSPCs to functional monocytes and macrophages.
  • HSPCs hematopoietic stem and/or hematopoietic progenitor cells
  • the culture method comprises three phases: (1) HSPC Expansion, comprised of culturing HSPCs with serum-free expansion medium in the presence of cytokines and growth factors, with or without activation of the Notch receptor signaling pathway by Deltal extI g G (DXI) binding, to induce proliferation of stem and progenitor cells without terminal differentiation; (2) Expansion and Monocyte Differentiation, comprised of culturing expanded HSPCs with serum-free medium in the presence of cytokines and growth factors, with or without activation of the Notch receptor signaling pathway by DXI binding, to induce further expansion and differentiation of cells to a monocyte phenotype; and (3) Macrophage Differentiation, comprised of culturing differentiated monocytes with medium containing serum or a serum replacement and growth factors to induce differentiation of monocytes to a macrophage phenotype.
  • HSPC Expansion comprised of culturing HSPCs with serum-free expansion medium in the presence of cytokines and growth factors
  • the hematopoietic stem cells, or stem and progenitor cells are CD34+.
  • the hematopoietic stem cells, or hematopoietic stem and progenitor cells are derived from human umbilical cord blood and/or human placental blood.
  • cord blood and/or placental blood derived CD34+ cells as the starting material, compared with adult-derived cells, allows for the generation of monocytes and macrophages that are not terminally differentiated or exhausted and are likely to have greater capacity for proliferation and persistence than adult monocytes derived from peripheral blood.
  • the CD34+ enriched cell population comprises hematopoietic stem or hematopoietic stem and progenitor cells and has been substantially depleted of T cells and red blood cells, therefore leaving enriched numbers of CD34+ hematopoietic stem or hematopoietic stem and progenitor cells.
  • the hematopoietic stem or hematopoietic stem and progenitor cells can in some embodiments comprise multiple HLA-types because the hematopoietic stem or hematopoietic stem and progenitor cells are not matched to each other prior to pooling, and also are not matched to the patient.
  • the HSPCs are either HLA matched or partially mismatched with the subject.
  • substantially depleted of T cells refers to less than 1 % CD3+ cells, or less than 0.5 % CD3+ cells, or less than 0.1 % CD3 + cells, in the enriched CD34+ cell population.
  • the CD34+ hematopoietic stem cells or hematopoietic stem and progenitor cells are derived from cord blood or from placental blood.
  • Human umbilical cord blood and/or human placental blood are typical sources of the cord blood stem cells.
  • Such blood can be obtained by methods known in the art. See, e.g., U.S. Patent Nos. 5,004,681 and 7,147,626 and U.S. Patent Publication No. 2013/0095079, incorporated herein by reference, for a discussion of collecting cord and placental blood at the birth of a human.
  • Umbilical cord blood and/or human placental blood collections are made under sterile conditions.
  • cord or placental blood is mixed with an anticoagulant, such as CPD (citrate-phosphate-dextrose), ACD (acid citrate-dextrose), Alsever’s solution (Alsever et al., 1941, N. Y. St. J. Med. 41: 126), De Gowin’s Solution (De Gowin, et al., 1940, J. Am. Med. Ass. 114:850), Edglugate-Mg (Smith, et al., 1959, J. Thorac. Cardiovasc. Surg. 38:573), Rous-Turner Solution (Rous and Turner, 1916, J. Exp. Med.
  • CPD citrate-phosphate-dextrose
  • ACD acid citrate-dextrose
  • Alsever s solution
  • De Gowin s Solution
  • Edglugate-Mg Smith, et al., 1959, J. Thorac. Cardiovasc. Surg. 38:573
  • Rous-Turner Solution Rous-Tur
  • Cord blood can preferably be obtained by direct drainage from the umbilical cord and/or by needle aspiration from the delivered placenta at the root and at distended veins.
  • the collected human cord blood and/or placental blood is free of contamination and, in particular, viral contamination.
  • the following tests can be performed on the collected blood, either routinely or where clinically indicated:
  • Bacterial culture To ensure the absence of microbial contamination, established assays can be performed, such as routine hospital cultures for bacteria under aerobic and anaerobic conditions.
  • Diagnostic screening for pathogenic microorganisms To ensure the absence of specific pathogenic microorganisms, various diagnostic tests can be employed. Diagnostic screening for any of the numerous pathogens transmissible through blood can be done by standard procedures. As one example, the collected blood sample (or a maternal blood sample) can be subjected to diagnostic screening for the presence of viruses. Any of numerous known assay systems can be used, based on the detection of virions, viral-encoded proteins, virus-specific nucleic acids, antibodies to viral proteins, and the like.
  • the collected blood can also be tested for infectious diseases, including but not limited to Human Immunodeficiency Virus-1 or 2 (HIV-1 or HIV-2), human T-Cell lymphotropic virus I and II (HTLV-I and HTLV-II), Hepatitis B, Hepatitis C, Cytomegalovirus, Syphilis, corona virus, West Nile Virus, and the like.
  • infectious diseases including but not limited to Human Immunodeficiency Virus-1 or 2 (HIV-1 or HIV-2), human T-Cell lymphotropic virus I and II (HTLV-I and HTLV-II), Hepatitis B, Hepatitis C, Cytomegalovirus, Syphilis, corona virus, West Nile Virus, and the like.
  • a maternal health history is determined to identify risks that the cord blood cells might pose, e.g., transmitting genetic or infectious diseases, such as cancer, leukemia, immune disorders, neurological disorders, hepatitis, or AIDS.
  • the collected cord blood can have undergone testing for one or more of cell viability, HLA typing, ABO/Rh typing, CD34+ cell count, and total nucleated cell count.
  • the blood is processed to produce an enriched hematopoietic stem cell population, or an enriched hematopoietic stem and progenitor cell population.
  • the hematopoietic stem cells, or hematopoietic stem and progenitor cells are CD34+ cells or predominantly CD34+ cells.
  • the hematopoietic stem cell or hematopoietic stem and progenitor cell population is substantially depleted of T cells and of red blood cells, resulting in a cell population enriched for CD34+ stem cells and/or CD34+ stem and progenitor cells.
  • Enrichment thus refers to a process wherein the percentage of hematopoietic stem cells, or hematopoietic stem and progenitor cells, in the cell population is increased (relative to the percentage in the population before the enrichment procedure). Purification is one example of enrichment.
  • a starting cord blood unit is made up of ⁇ 0.25 % - 0.75 % CD34+ cells
  • the enriched selected cell population is made up of ⁇ 92 % - 99 % CD34+ cells.
  • the collected cord and/or placental blood can be fresh or have been previously cryopreserved.
  • Any suitable technique known in the art for cell separation/selection can be used to carry out the enrichment for hematopoietic stem cells, or hematopoietic stem and progenitor cells. Methods which rely on differential expression of cell surface markers can be used. For example, cells expressing the cell surface marker CD34 can be positively selected using a monoclonal antibody to CD34, such that cells expressing CD34 are retained, and cells not expressing CD34 are not retained.
  • the separation techniques employed should maximize the viability of the cell population to be selected. The particular technique employed will depend upon the efficiency of separation, cytotoxicity of the methodology, ease and speed of performance, and the necessity for sophisticated equipment and/or technical skill.
  • Procedures for separation can include magnetic separation, using antibody-coated magnetic beads, affinity chromatography, and “panning” with antibody attached to a solid matrix, e.g., plate, or other convenient technique.
  • Techniques providing accurate separation/selection include fluorescence activated cell sorters, which can have varying degrees of sophistication, e.g., a plurality of color channels, low angle and obtuse light scattering detecting channels, impedance channels, and the like.
  • the antibodies used in the selection process may be conjugated with markers, such as magnetic beads, which allow for direct separation, biotin, which can be removed with avidin or streptavidin bound to a support, fluorochromes, which can be used with a fluorescence activated cell sorter, or the like, to allow for ease of separation of the particular cell type.
  • markers such as magnetic beads, which allow for direct separation, biotin, which can be removed with avidin or streptavidin bound to a support, fluorochromes, which can be used with a fluorescence activated cell sorter, or the like, to allow for ease of separation of the particular cell type.
  • markers such as magnetic beads, which allow for direct separation, biotin, which can be removed with avidin or streptavidin bound to a support, fluorochromes, which can be used with a fluorescence activated cell sorter, or the like, to allow for ease of separation of the particular cell type.
  • Any technique may be employed which is not unduly detrimental to the viability of the remaining
  • fresh cord blood units are processed to select for, i.e., enrich for, CD34+ cells using anti-CD34 antibodies directly or indirectly conjugated to magnetic particles in connection with a magnetic cell separator, for example, the CliniMACS® Cell Separation System (Miltenyi Biotec, Bergisch Gladbach, Germany), which employs nano-sized super-paramagnetic particles composed of iron oxide and dextran coupled to specific monoclonal antibodies.
  • a magnetic cell separator for example, the CliniMACS® Cell Separation System (Miltenyi Biotec, Bergisch Gladbach, Germany), which employs nano-sized super-paramagnetic particles composed of iron oxide and dextran coupled to specific monoclonal antibodies.
  • the CliniMACS® Cell Separator is a closed sterile system, outfitted with a single-use disposable tubing set. The disposable tubing set can be used for and discarded after processing a single unit of collected cord and/or placental blood to enrich for CD34+ cells.
  • two or more umbilical cord blood and/or placental blood units can be pooled prior to enriching for the hematopoietic stem cells, or hematopoietic stem and progenitor cells.
  • individual populations of CD34+ stem cells or CD34+ stem and progenitor cells can be pooled after enriching for the hematopoietic stem cells, or hematopoietic stem and progenitor cells.
  • the number of umbilical cord blood and/or placental blood units, or populations of hematopoietic stem or hematopoietic stem and progenitor cells that are pooled is 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or at least any of the foregoing numbers.
  • the pool contains 2 to 8, 2 to 10, 4 to 8, 4 to 10, 2 to 20, 4 to 20, 2 to 25 or 4 to 25, and no more than 20 or 25, umbilical cord blood and/or placental blood units, or CD34+ hematopoietic stem or hematopoietic stem and progenitor cell populations.
  • the umbilical cord blood and/or placental blood units or hematopoietic stem or hematopoietic stem and progenitor cell populations are pooled without regard to the HLA-type of the hematopoietic stem or hematopoietic stem and progenitor cells.
  • the cells in the pool are derived from the umbilical cord blood and/or placental blood of individuals of the same race, e.g., African- American, Caucasian, Asian, Hispanic, Native- American, Australian Europe Europe, Inuit, Pacific Islander, or derived from umbilical cord blood and/or placental blood of individuals of the same ethnicity, e.g., Irish, Italian, Indian, Japanese, Chinese, Russian, and the like.
  • the cells in the pool are combined without regard to race or ethnicity.
  • the red blood cells and white blood cells of the cord blood or placental blood can be separated.
  • the red blood cell fraction can be discarded, and the white blood cell fraction can be processed in the magnetic cell separator as described above to enrich for CD34+ hematopoietic stem cells or hematopoietic stem and progenitor cells. Separation of the white and red blood cell fractions can be performed by any method known in the art, including centrifugation techniques.
  • FICOLL- PAQUETM is normally placed at the bottom of a conical tube, and the whole blood is layered above. After being centrifuged, the following layers will be visible in the conical tube, from top to bottom: plasma and other constituents, a layer of mono-nuclear cells called buffy coat containing the peripheral blood mononuclear cells (white blood cells), FICOLL-PAQUETM, and erythrocytes and granulocytes, which should be present in pellet form.
  • This separation technique allows easy harvest of the peripheral blood mononuclear cells (PBMCs).
  • an aliquot of the cord blood or placental unit can be checked for total nucleated cell count and/or CD34+ cell content.
  • both CD34+ and CD34- cell fractions are recovered.
  • DNA can be extracted from a sample of the CD34- cell fraction for initial HLA typing and future chimerism studies, even though HLA matching of the CD34+ cell fraction to the patient or to the other cord blood or placental blood units is not done.
  • HSPCs Phase 1 - Expansion of Hematopoietic Stem and Progenitor Cells
  • “Expanded HSPCs” refers to hematopoietic stem cells or stem and progenitor cells that have been subjected to a technique for expanding the hematopoietic stem cells, or hematopoietic stem and progenitor cells ex vivo, which technique has been shown to result in (i) an increase in the number of hematopoietic stem cells, or hematopoietic stem and progenitor cells, in an aliquot of the cells thus expanded, or (ii) an increased number of severe combined immunodeficiency (SCID) repopulating cells determined by limiting-dilution analysis as shown by enhanced engraftment in non-obese diabetic (NOD)/SCID mice infused with an aliquot of the cells thus expanded.
  • SCID severe combined immunodeficiency
  • the umbilical cord blood and/or placental blood units are red cell depleted, and the number of CD34+ cells in the red cell depleted fraction is determined.
  • the umbilical cord blood and/or placental blood samples enriched for CD34+ cells are seeded into tissue culture treated plastic culture vessels lacking any additional coating at a density of 9,000 to 10,000 cells/cm 2 .
  • the hematopoietic stem cells or hematopoietic stem and progenitor cells have been isolated (e.g., from human cord blood and/or human placental blood collected from humans at birth) according to the enrichment methods described above or other methods known in the art, the hematopoietic stem cells or hematopoietic stem and progenitor cells are expanded to increase the number of hematopoietic stem cells or hematopoietic stem and progenitor cells, e.g., CD34+ cells.
  • Any method known in the art for expanding the number of hematopoietic stem cells or hematopoietic stem and progenitor cells that gives rise to an expanded (z.e., increased number of) population of hematopoietic stem cells or hematopoietic stem and progenitor cells can be used.
  • the hematopoietic stem cells or hematopoietic stem and progenitor cells are cultured under cell growth conditions (e.g., promoting mitosis) such that the hematopoietic stem cells or hematopoietic stem and progenitor cells grow and divide (proliferate) to obtain an expanded population of CD34+ hematopoietic stem cells or hematopoietic stem and progenitor cells.
  • cell growth conditions e.g., promoting mitosis
  • individual populations of hematopoietic stem cells or hematopoietic stem and progenitor cells derived from an umbilical cord blood and/or placental blood of a single human at birth can be pooled, without matching to the HLA type of the other hematopoietic stem cells or hematopoietic stem and progenitor cells, prior to or after expansion.
  • the hematopoietic stem cells or hematopoietic stem and progenitor cells are expanded prior to pooling.
  • the technique used for expansion is one that has been shown to (i) result in an increase in the number of hematopoietic stem cells, or hematopoietic stem and progenitor cells, e.g., CD34+ cells, in the expanded stem cell product relative to the unexpanded population of hematopoietic stem cells or stem and progenitor cells, where the unexpanded cell population and expanded cell population are from different aliquots of the same source of stem or stem and progenitor cells, wherein the expanded cells but not the unexpanded cells are subjected to the expansion technique.
  • hematopoietic stem cells or hematopoietic stem and progenitor cells, e.g., CD34+ cells
  • Expansion techniques include but are not limited to those described in U.S. Patent No. 7,399,633 B2; U.S. Patent Publication No. 2013/0095079; Delaney etal., 2010, Nature Med. 16(2): 232-236; Zhang et al., 2008, Blood 111:3415-3423; or Himburg et al., 2010, Nature Medicine 16(4):475-82, each incorporated herein by reference, as well as those described below.
  • the hematopoietic stems cells or hematopoietic stem and progenitor cells are cultured in culture medium in the presence of growth factors, and are exposed to cell growth conditions (e.g., promoting mitosis) such that the hematopoietic stem or hematopoietic stem and progenitor cells proliferate to obtain an expanded population of hematopoietic stem or hematopoietic stem and progenitor cells.
  • cell growth conditions e.g., promoting mitosis
  • the hematopoietic stem or hematopoietic stem and progenitor cells are exposed to cell growth conditions (e.g., promoting mitosis) such that the hematopoietic stem or hematopoietic stem and progenitor cells proliferate to generate an expanded hematopoietic stem or hematopoietic stem and progenitor cell population.
  • cell growth conditions e.g., promoting mitosis
  • the increase in the number of CD34+ cells (or other suitable antigen-positive cells) as a percentage of cells in the expanded stem cell product, relative to the population prior to the enrichment procedure is at least 25-, 50-, 75-, 100-, 150-, 200-, 250-, 300-, 350-, 400- or at least 350-fold, and preferably is 100- to 200-fold or 100- to 400-fold.
  • the expanded hematopoietic stem or hematopoietic stem and progenitor cell population so obtained can be frozen and stored for later use.
  • the hematopoietic stem or hematopoietic stem and progenitor cells are cultured for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 days or more; or, preferably, the hematopoietic stem or hematopoietic stem and progenitor cells are cultured for 3, 7, 10, 14, or 21 days. In more preferred embodiments, the cells are expanded for at least 10 days or from about 14 to about 21 days.
  • An exemplary culture condition for expanding the hematopoietic stem or hematopoietic stem and progenitor cells includes culturing the cells for about 14 to 21 days in the presence of a serum free medium supplemented with the following human growth factors: stem cell factor, Flt-3 receptor ligand, Thrombopoietin, Interleukin-6 and Interleukin-3.
  • the foregoing growth factors are present at the following concentrations: 50 to 300 ng/mL stem cell factor, 50 to 300 ng/mL Flt-3 receptor ligand, 50 to 100 ng/mL Thrombopoietin, 50 to 100 ng/mL Interleukin-6 and 10 ng/mL Interleukin-3.
  • the cell culture medium consists of X- VIVOTM- 10 (Lonza).
  • the base medium used during HSPC expansion culture can be StemSpanTM SFEM II, StemProTM-34 SFM, X- VIVOTM- 15, PRIME-XV Expansion XSFM, CellGenix® SCGM, StemLine® or StemLine® II Hematopoietic Stem Cell Expansion Medium, or StemMACS HSC Expansion Media.
  • Monocytes are approximately 0.10 pm diameter round mononuclear white blood cells that travel from bone marrow through the peripheral blood to tissues where they can further differentiate into macrophages or dendritic cells.
  • stimulated monocytes demonstrate properties of chemotaxis and phagocytosis and can secrete cytokines that attract and/or activate additional cells of the innate and adaptive immune system.
  • Cytokines released by monocytes include, but are not limited to, tumor necrosis factor alpha (TNFa), interleukin 6 (IL-6), interleukin 12 (IL- 12), and interleukin 15 (IL-15).
  • Macrophages are large (approximately 21 pm diameter), complex, adherent, vacuolated white blood cells derived from monocytes, and are located mostly within tissues, where they can act as phagocytic scavengers to clear cellular debris related to cell aging or wound healing, can digest and present pathogen-related antigens to other immune cells to initiate an immune response, and can secrete powerful cytokines involved in inflammation and the regulation of immune responses. Tissue resident macrophages often perform functions specific to the tissue in which they reside but are generally classified into simplified groupings based on their broad functional and activation status, markers expressed on the cell surface, and cytokines released.
  • the “classically activated” Ml polarized macrophages are pro-inflammatory, anti-pathogenic cells that express surface markers including, but not limited to, CD40, CD80, CD86, and interferon gamma (IFNy) receptor, and can secrete cytokines including, but not limited to, TNFa, IL- 12, and IL-6.
  • the “alternatively activated” M2 polarized macrophages are anti-inflammatory and involved in tissue remodeling and immune regulation, express the surface markers including, but not limited to, CD 163, CD206, CD209, and interleukin 4 (IL-4) receptor alpha, and can secrete cytokines including, but not limited to, interleukin 10 (IL- 10) and transforming growth factor beta (TGFP).
  • Tumor-associated macrophages are identified within a tumor microenvironment and are often influenced by that microenvironment to adopt an immunosuppressive phenotype.
  • Phase 1 the HSPC Expansion culture
  • cells were harvested and washed with, for example, phosphate buffered saline (PBS) by centrifugation, and resuspended in Phase 2 culture medium.
  • the base cell culture medium for this phase can be selected from, for example, StemSpanTM SFEM II, StemProTM-34 SFM, X- VIVOTM- 10, X- VIVOTM- 15, or IMDM.
  • the base cell culture medium is X- VIVOTM- 10 medium (Lonza).
  • the expanded HSPCs are also contacted with human growth factors that can continue to expand the population of HSPCs as in Phase 1, and also include human cytokines that induce the differentiation of the expanded HSPCs into Monocytes.
  • the human growth factors and/or cytokines can include monocyte colony stimulating factor (M-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), Flt3-L, IL-6 and/or IL-3.
  • An exemplary culture condition for further expanding the hematopoietic stem or hematopoietic stem and progenitor cells and inducing the differentiation of the expanded HSPCs into monocytes includes culturing the cells for about 7, 10, or 14 days in the presence of a serum free medium supplemented with the following human growth factors: M-CSF (50 ng/mL), Fit- 3 receptor ligand (Flt3-L; 50 ng/mL), IL-3 (10 ng/mL), and IL-6 (50 ng/mL).
  • M-CSF 50 ng/mL
  • Fit- 3 receptor ligand Flt3-L
  • IL-3 10 ng/mL
  • IL-6 50 ng/mL
  • the Expansion and monocyte differentiation media supplements can include a combination of 50 ng/mL MCSF, 50 ng/mL GM-CSF, 50 ng/mL Flt3L, and 10 ng/mL IL-3.
  • the Monocyte cell product produced can be cryopreserved for later use or used directly in Phase 3 - Macrophage Differentiation.
  • the monocyte cell product can also be formulated as a pharmaceutical product.
  • Phase 2 Extracellular and Monocyte Differentiation culture
  • cells were harvested and washed with PBS by centrifugation, and resuspended in Phase 3 culture medium supplemented with human growth factors and other factors that can induce to differentiation of monocytes into macrophage.
  • cells cryopreserved at the end of Phase 2 can be thawed and resuspended in Phase 3 culture medium.
  • Phase 2 cells enriched for monocytes were seeded into tissue culture treated plastic culture vessels lacking any additional coating at a density of about 130,000 to 230,000 cells/cm 2 .
  • Cells were cultured for 6 to 8 days with a feed with Phase 3 medium about 3 to about 4 days into the culture period.
  • the cells are cultured for about 7 days.
  • non-adherent cells are collected with the culture medium into a sterile conical tube.
  • the remaining adherent cells are incubated to loosen them from the tissue culture substrate with, for example, TrypLETM Express Enzyme (Gibco), followed by scraping of the tissue culture plastic surface, and the released adherent cells are combined with the collected non-adherent cells and washed by centrifugation.
  • Washed cells can be used for in vitro or in vivo analyses, formulation into a pharmaceutical cell product, and/or cryopreserved.
  • the base cell culture medium can be selected from X-VIVOTM-10, X-VIV0TM-15, RPMI 1640, IMDM, or ImmunoCultTM-SF Macrophage Medium.
  • the base cell culture medium is RPMI 1640.
  • the base cell culture medium is also typically supplemented with serum or a serum replacement.
  • the serum or serum replacement can be fetal bovine serum (FBS), human AB serum, human platelet lysate (HPL), and/or human serum albumin (HSA).
  • the serum or serum replacement is about 10 % FBS; or about 10 % human AB serum; or about 2.5 % HPL, 5% HPL, or 10% HPL; or about 0.25 % HSA, 0.5% HSA, 1% HSA, or 2% HSA.
  • the base medium is also supplemented with certain human growth factors and/or cytokines to induce the differentiation of the monocytes into macrophage.
  • the human growth factors and/or cytokines can be, for example, GM-CSF and/or M-CSF.
  • about 10 ng/mL, 25 ng/mL, or 50 ng/mL of GM-CSF can be used.
  • about 25 ng/mL, 50 ng/mL, or 100 ng/mL M-CSF can be used.
  • the base culture media is RPMI 1640, supplemented with 10 ng/mL GM-CSF and either 2.5 % HPL or 10 % FBS.
  • the macrophage cell product produced at the end of Phase 3 can be cryopreserved using, for example, CryoStor® CS 10 (Biolife Solutions) for later use or used directly to formulate a pharmaceutical product.
  • CryoStor® CS 10 Biolife Solutions
  • In vitro culture of differentiating cells with feeder cell layers or with a tissue culture vessel substrate or coating can be done to activate cell signaling pathways that influence cell differentiation toward a specific desired lineage or activation status.
  • cells have been cultured in tissue culture treated plastic culture vessels without additional coating or pre-treatment.
  • the enriched HSPCs have been cultured in a tissue culture substrate pre-coated with a Notch ligand and a portion of fibronectin as used in well-known methods for HSPC expansion.
  • DXI Deltal extIgG
  • RetroNectin® 0.8 pg/cm 2 recombinant human fibronectin fragment
  • cells were cultured in Phase 2 Expansion and Monocyte Differentiation culture, in tissue culture treated plastic culture vessels without additional coating or pre-treatment; pre-coated with 0.1 pg/cm 2 DXI and 0.8 pg/cm 2 RetroNectin®, with 0.2 pg/cm 2 DXI and 0.8 pg/cm 2 RetroNectin®, and with 0.4 pg/cm 2 DXI and 0.8 pg/cm 2 RetroNectin®.
  • the cells were then cultured in Phase 3 Macrophage Differentiation in tissue culture treated plastic culture vessels without additional coating or pre-treatment. These methods all resulted in the production of a monocyte and/or macrophage product, although the yields were not better than those methods where Notch ligand was not used.
  • the monocytes/macrophage cell product generated through these methods can be genetically modified during any culture phase during their expansion or differentiation through standard methods of cell engineering that can include viral transduction with lentiviral, retroviral, adenoviral, or adeno-associated viral (AAV) vectors, or electroporation of mRNA, short hairpin RNA (shRNA), silencing RNA (siRNA), or guide RNA (gRNA), or plasmid DNA or covalently closed DNA or transposons or enzymes, or combinations of these materials, or lipid fusion with cell membranes to deliver mRNA, shRNA, siRNA, gRNA, or plasmid DNA or covalently closed DNA or transposons or enzymes, or combinations of these materials encapsulated within simple lipids or lipid nanoparticles.
  • the genetic modification can be integrated within the genome of the cell and present through the lifespan of the cell, or the genetic modification may not be integrated within the genome of the cell and be only temporarily present within the
  • Such cell engineering can be carried out for the purposes of prolonging the lifespan of the modified cells, enhancing endogenous functions of the modified cells, stabilizing desired phenotypes of the modified cells, introducing new capabilities or functions to the modified cells, or combinations of the above.
  • modifications could include expression of a receptor (such as a CAR, a TCR, a chimeric receptor or other receptor), an immune cell engager, an antibody, a nanobody, a transcription factor, a cytokine, and/or a chemokine.
  • Such modifications can also include knockdown, knockout, and/or knock-in of a gene or multiple genes.
  • Another potential modification can include pre-loading macrophages with immune activating cargo, such as an oncolytic virus or other immunomodulatory molecule, for delivery to the otherwise immunosuppressive solid tumor microenvironment.
  • the monocyte/macrophage product is engineered to have one or more gene knockdowns, knockouts, or knock-ins.
  • the gene knockdown is achieved by the use of shRNAs or siRNAs.
  • the gene knockdown, knockout, or knock-in is achieved by targeted enzymatic cleavage followed by homology-directed repair or non-homologous end joining.
  • the enzyme can include but is not limited to Cas enzymes (Cas 7-11, Cas9, dCas9, nCas9, Casl2a), zinc-finger nucleases (ZFNs), or transcription activator-like effector nucleases (TALENs).
  • the enzyme is fused to one or more activating or repressor effector domains such as Kruppel-associated box (KRAB) domain, VP64, Rta, p65, or HSF1.
  • KRAB Kruppel-associated box
  • targeted enzymatic cleavage is achieved using nucleic acids such as guide RNAs (gRNAs).
  • gene knockdown, knockout, or knock-in is achieved through transient expression of enzymes and/or nucleic acid(s).
  • gene knockdown, knockout, or knock-in is achieved through sustained expression of enzymes and/or nucleic acid(s).
  • the enzymes and nucleic acid(s) are delivered via transfection, electroporation, or viral transduction.
  • plasmids or viral vectors encode the nucleic acid sequences required for shRNAs, siRNAs, gRNAs, enzymes, or the transgene for targeted gene knock-in.
  • the gene targeted for knockdown or knockout includes, but is not limited to beta-2-microglobulin (B2M) or class II major histocompatibility complex transactivator (CIITA).
  • the gene targeted for knockdown or knockout includes a cytokine, a cytokine receptor, a chemokine, a chemokine receptor, a transcription factor, a growth factor receptor, an immunomodulatory molecule, a stimulatory molecule, a costimulatory molecule, and/or a costimulatory ligand.
  • the gene that is knocked-in encodes, for example, HLA-E or HLA-E and B2M.
  • the gene that is knocked-in encodes a cytokine, a cytokine receptor, a chemokine, a chemokine receptor, a transcription factor, a growth factor receptor, an immunomodulatory molecule, a stimulatory molecule, a costimulatory molecule, and/or a costimulatory ligand.
  • the transgene that is knocked-in is a CAR or Non-CAR construct as described below.
  • the monocyte/macrophage product is genetically modified to express a CAR.
  • a CAR can comprise an extracellular antigen-binding domain, a spacer (or hinge) region, a transmembrane domain, and one or more intracellular signaling domains, wherein the extracellular antigen-binding domain specifically binds to an antigen, e.g., a tumor antigen or a pathogen antigen, including for example a viral or bacterial antigen.
  • Second generation CARs are typically composed of an extracellular antigen binding domain (e.g., a single-chain variable fragment (scFv)) fused to a transmembrane domain, fused to cytoplasmic/intracellular signaling domain of the T cell receptor chain.
  • First generation CARs typically have the intracellular signaling domain from the CD3zeta-chain, which is the primary transmitter of signals from endogenous TCRs.
  • First generation” CARs can provide de novo antigen recognition and cause activation of cells through their CD3zeta chain signaling domain in a single fusion molecule, independent of HLA-mediated antigen presentation.
  • “Second generation” CARs add intracellular signaling domains from various co- stimulatory molecules (e.g., CD28, 4-1BB, ICOS, OX40, or 2B4) to the cytoplasmic tail of the CAR to provide additional signals to the cell.
  • “Second generation” CARs comprise those that provide both co- stimulation (e.g., CD28, 4- IBB or 2B4) and activation (CD3zeta). Preclinical studies have indicated that “Second Generation” CARs can improve the anti-tumor activity of T cells.
  • “Second Generation” CAR modified T cells were demonstrated in clinical trials targeting the CD 19 molecule in patients with chronic lymphoblastic leukemia (CLL) and acute lymphoblastic leukemia (ALL).
  • “Third generation” CARs comprise those that provide multiple co-stimulation (e.g., CD28 and 4- IBB) and activation (CD3zeta).
  • the extracellular antigen-binding domain of the CAR (embodied, for example, as an scFv or an analog thereof) binds to an antigen with a dissociation constant (IQ) of about 2 xlO -7 M or less.
  • IQ dissociation constant
  • the BQ is about 1 x IO’ 7 M or less, about 5 x 10’ 8 M or less, about 1 x 10’ 8 M or less, about 5 x 10’ 9 M or less, or about 1 x 10’ 9 M or less.
  • Binding of an extracellular antigen-binding domain (for example, an scFv or an analog thereof) of an antigen-targeted CAR can be confirmed by, for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growth inhibition), or Western Blot assay.
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • FACS analysis e.g., FACS analysis
  • bioassay e.g., growth inhibition
  • Western Blot assay Western Blot assay.
  • Each of these assays generally detect the presence of protein- antibody complexes of particular interest by employing a labeled reagent (e.g., an antibody, or an scFv) specific for the complex of interest.
  • a labeled reagent e.g., an antibody, or an scFv
  • the scFv can be radioactively labeled and used in a radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March 1986, which is incorporated by reference herein).
  • the radioactive isotope can be detected by such means as the use of a gamma counter or a scintillation counter or by autoradiography.
  • the extracellular antigen-binding domain of the CAR is labeled with a fluorescent marker.
  • Non-limiting examples of fluorescent markers include green fluorescent protein (GFP), blue fluorescent protein (e.g., EBFP, EBFP2, Azurite, and mKalamal), cyan fluorescent protein (e.g., ECFP, Cerulean, and CyPet), and yellow fluorescent protein (e.g., YFP, Citrine, Venus, and YPet).
  • GFP green fluorescent protein
  • blue fluorescent protein e.g., EBFP, EBFP2, Azurite, and mKalamal
  • cyan fluorescent protein e.g., ECFP, Cerulean, and CyPet
  • yellow fluorescent protein e.g., YFP, Citrine, Venus, and YPet
  • the extracellular antigen-binding domain, the spacer region, or the intracellular signaling domain of the CAR is labeled with an epitope tag.
  • epitope tags include hemagglutinin (HA), c-Myc, FLAG, 6-His, and
  • the extracellular antigen-binding domain specifically binds to an antigen.
  • the extracellular antigen-binding domain is an scFv.
  • the scFv is a human scFv.
  • the scFv is a humanized scFv.
  • the extracellular antigen-binding domain is a Fab, which is optionally crosslinked.
  • the extracellular binding domain is a F(ab’)2- I n certain embodiments, any of the foregoing molecules may be comprised in a fusion protein with a heterologous sequence to form the extracellular antigen-binding domain.
  • the scFv is identified by screening scFv phage library with an antigen-Fc fusion protein.
  • the antigen is a tumor antigen.
  • the antigen is a pathogen antigen, including for example, a viral or a bacterial antigen.
  • the extracellular binding domain is an scFv that specifically binds to a tumor- specific, tumor-associated, or stroma antigen.
  • the tumor- specific, tumor-associated, or stroma antigen can be for example, AFP, ALPP, ALPP2, ANTXR1, alpha-V beta-3 integrin, alpha-V beta-6 integrin, AXL, BCMA, B7-H3 (CD276), B7-H4 (VTCN1), carbonic anhydrase IX (CA1X), carcinoembryonic antigen (CEA), CD5, CD8, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD44V6, CD47, CD49c, CD49e, CD49f, CD56, CD61, CD66c, CD70, CD72, CD73, CD74, CD80, CD86, CD104, CD123, CD126, CD133, CD138, CD142, CD147, CD318,
  • the tumor specific target antigen is CD 19, such as an scFv derived from the FMC63 antibody or 4G7 antibody.
  • the scFv comprises the CDRs of the FMC63 antibody: a CDRL1 sequence of RASQDISKYLN (SEQ ID NO: 1), a CDRL2 sequence of SRLHSGV (SEQ ID NOG), a CDRL3 sequence of GNTLPYTFG (SEQ ID NOG), a CDRH1 sequence of DYGVS (SEQ ID NOG), a CDRH2 sequence of VTWGSETTYYNSALKS (SEQ ID NOG), and a CDRH3 sequence of YAMDYWG (SEQ ID NOG); or a CDRL1 sequence of RASQDISKYLN (SEQ ID NO: 1), a CDRL2 sequence of SRLHSGV (SEQ ID NOG), a CDRL3 sequence of GNTLPYTFG (SEQ ID NOG), a CDRH1 sequence of a CDRL2 sequence of SRLHSGV
  • an extracellular binding domain is an scFv derived from or comprising the heavy and light chain variable regions of antibody FMC63.
  • the heavy and light chain variable regions of antibody FMC63 are shown in SEQ ID NO:64 and SEQ ID NO:65, respectively.
  • SEQ ID NO:64 FMC63 heavy chain variable region EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGV IWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYY YGGSYAMDYWGQGTSVTVSS
  • SEQ ID NO:65 FMC63 light chain variable region DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHS GVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT
  • an scFv comprises the heavy and light chain CDRs of the CD 19 monoclonal antibody 4G7, as set forth in SEQ ID NO:25 and SEQ ID NO:26 or SEQ ID NO:27.
  • the extracellular antigen binding comprises the heavy and light chain variable region CDRs set forth in SEQ ID NO:25 and 26, or in SEQ NO:25 and 27.
  • an scFv is derived from the CD 19 monoclonal antibody 4G7, preferably comprises a part of the binding domains of CD 19 monoclonal antibody 4G7, portions of variable region of the CD 19 monoclonal antibody 4G7 immunoglobulin gamma 1 heavy chain (SEQ ID NO:25) and the variable fragments of the CD19 monoclonal antibody 4G7 immunoglobulin kappa light chain (SEQ ID NO:26 or SEQ ID NO:27) linked together by a flexible linker.
  • the flexible linker has the amino acid sequence set forth in SEQ ID NO:28.
  • the extracellular antigen binding comprises the heavy and light chain variable region CDRs set forth in SEQ ID NO:25 and SEQ ID NO:26, or in SEQ NO:25 and SEQ ID NO:27.
  • SEQ ID NO:25 anti-human CD19 monoclonal antibody 4G7 heavy chain variable region EVQLQQSGPELIKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYINPY NDGTKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARGTYYYGSRVF DYWGQGTTLTVSS
  • an scFv is derived from a CLA (cutaneous lymphocyte antigen) monoclonal antibody and comprises the heavy and light chain variable region of the CLA monoclonal antibody set forth in SEQ ID NO:29 and SEQ ID NO:30, respectively (see US Patent Application Publication No. 2019/0209611; incorporated herein by reference).
  • the extracellular antigen binding domain comprises the heavy and light chain variable region CDRs set forth in SEQ ID NO:29 and SEQ ID NO:30.
  • an scFv is derived from a CD 142 monoclonal antibody and comprises the heavy and light chain variable region of the CD 142 monoclonal antibody set forth in SEQ ID NOs:31 and 32, respectively (see US Patent Application Publication No. 2019/0209611, incorporated herein by reference).
  • the extracellular antigen binding domain comprises the heavy and light chain variable region CDRs set forth in SEQ ID NO:31 and SEQ ID NO:32.
  • an scFv is derived from a CD73 monoclonal antibody and comprises the heavy and light chain variable region of the CD73 monoclonal antibody set forth in SEQ ID NOs:33 and 34, respectively (see US Patent Application Publication No. 2019/0209611, incorporated herein by reference).
  • the extracellular antigen binding domain comprises the heavy and light chain variable region CDRs set forth in SEQ ID NO:33 and SEQ ID NO:34.
  • an scFv is derived from a CD49c monoclonal antibody and comprises the heavy and light chain variable region of the CD49c monoclonal antibody set forth in SEQ ID NOs:35 and 36, respectively (see US Patent Application Publication No. 2019/0209611; incorporated herein by reference).
  • the extracellular antigen binding domain comprises the heavy and light chain variable region CDRs set forth in SEQ ID NO:35 and SEQ ID NO:36.
  • an scFv is derived from a CD66c monoclonal antibody and comprises the heavy and light chain variable region of the CD66c monoclonal antibody set forth in SEQ ID NOs:37 and 38, respectively (see US Patent Application Publication No. 2019/0209611, incorporated herein by reference).
  • the extracellular antigen binding domain comprises the heavy and light chain variable region CDRs set forth in SEQ ID NO:37 and SEQ ID NO:38.
  • an scFv is derived from a CD 104 monoclonal antibody and comprises the heavy and light chain variable region of the CD 104 monoclonal antibody set forth in SEQ ID NOs:39 and 40, respectively (see US Patent Application Publication No. 2019/0209611; incorporated herein by reference).
  • the extracellular antigen binding domain comprises the heavy and light chain variable region CDRs set forth in SEQ ID NO:39 and SEQ ID NO:40.
  • CD104 VH (heavy chain variable region) QVNLLQSGAALVKPGASVKLSCKASGYTFTDYYIFWVKQSHGKSLEWIGYINPN SGSTNYNEKFKRKATLSVDKSTNTAYMELSRLTSEDSATYYCTRRAYYGYNPFD YWGQGVMVTVSS
  • CD 104 VL (light chain variable region) DIQMTQTPSSMPASLGERVTISCRASRGINNYLSWYQQNLDGTIKPLIYYTSNLQS GVPSRFSGSGSGTDYSLTISSLEPEDFAMYYCQQYDSSPWTFGGGTKLELK
  • an scFv is derived from a CD318 monoclonal antibody and comprises the heavy and light chain variable region of the CD318 monoclonal antibody set forth in SEQ ID NO:41 and SEQ ID NO:42, respectively (see US Patent Application Publication No. 2019/0209611; incorporated herein by reference).
  • the extracellular antigen binding domain comprises the heavy and light chain variable region CDRs set forth in SEQ ID NO:41 and SEQ ID NO:42.
  • an scFv is derived from a TSPAN8 monoclonal antibody and comprises the heavy and light chain variable region of the TSPAN8 monoclonal antibody set forth in SEQ ID NOs:43 and 44, respectively (see US Patent Application Publication No. 2019/0209611; incorporated herein by reference).
  • the extracellular antigen binding domain comprises the heavy and light chain variable region CDRs set forth in SEQ ID NO:43 and SEQ ID NO:44.
  • an scFv to CLA has the amino acid sequence set forth in SEQ ID NO:45 or SEQ ID NO:46 (see US Patent Application Publication No. 2019/0209611; incorporated herein by reference).
  • an scFv to CD 142 has the amino acid sequence set forth in
  • an scFv to CD73 has the amino acid sequence set forth in SEQ ID NO:49 or SEQ ID NO:50 (see US Patent Application Publication No. 2019/0209611; incorporated herein by reference).
  • an scFv to CD49c has the amino acid sequence set forth in SEQ ID NO:51 or SEQ ID NO:52 (see US Patent Application Publication No. 2019/0209611; incorporated herein by reference).
  • an scFv to CD66c has the amino acid sequence set forth in SEQ ID NO:53 or SEQ ID NO:54 (see US Patent Application Publication No. 2019/0209611; incorporated herein by reference).
  • an scFv to CD 104 has the amino acid sequence set forth in SEQ ID NO:55 or SEQ ID NO:56 (see US Patent Application Publication No. 2019/0209611; incorporated herein by reference).
  • an scFv to CD318 has the amino acid sequence set forth in SEQ ID NO:57 or SEQ ID NO:58 (see US Patent Application Publication No. 2019/0209611; incorporated herein by reference).
  • an scFv to TSPAN8 has the amino acid sequence set forth in SEQ ID NO:59 or SEQ ID NO:60 (see US Patent Application Publication No. 2019/0209611; incorporated herein by reference).
  • the transmembrane domain of the CAR comprises a hydrophobic alpha helix that spans at least a portion of the membrane. Different transmembrane domains result in different receptor stability. After antigen recognition, receptors cluster and a signal is transmitted to the cell.
  • the transmembrane domain of the CAR can comprise, for example, a CD8 polypeptide, a CD28 polypeptide, a NKG2D polypeptide, a CD3zeta polypeptide, a CD4 polypeptide, a 4- IBB polypeptide, an OX40 polypeptide, an ICOS polypeptide, a synthetic peptide (not based on a protein associated with the immune response), or a combination thereof.
  • the transmembrane domain of a CAR comprises the transmembrane domain of the molecule, or portion of the molecule, used in the membrane proximal region of the intracellular signaling domain of the CAR.
  • the transmembrane domain comprises a CD8 polypeptide.
  • the CD8 polypeptide has an amino acid sequence that is at least about 85 %, about 90 %, about 95 %, about 96 %, about 97 %, about 98 %, about 99 % or about 100 % identical to the sequence having a NCBI Reference No: NP_001139345.1, SEQ ID NO:7, (sequence identity herein may be determined using standard software such as BLAST or FASTA), or fragments thereof, and/or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions.
  • a “conservative amino acid substitution” means an amino acid substitution wherein an amino acid is substituted for another electronically similar amino acid.
  • an amino acid with a hydrophobic side chain can be substituted for a different amino acid also having a hydrophobic side chain (e.g., leucine substituted for isoleucine, alanine substituted for valine, and the like); an amino acid with an acidic chain can be substituted for a different amino acid also having an acidic side chain (e.g., aspartic acid substituted for glutamic acid, and the like); an amino acid with a basic chain can be substituted for a different amino acid also having a basic side chain (e.g., lysine substituted for arginine, and the like); and an amino acid with a polar side chain can be substituted for a different amino acid also having a polar side chain (e.g., serine substituted for threonine, and the like).
  • the CD8 polypeptide can have an amino acid sequence that is a consecutive portion of SEQ ID NO:7 which is at least 20, or at least 30, or at least 40, or at least 50, and up to 235 amino acids in length.
  • the CD8 polypeptide comprises or has an amino acid sequence of amino acids 1 to 235, 1 to 50, 50 to 100, 100 to 150, 150 to 200, or 200 to 235 of SEQ ID NO:7.
  • a CAR comprises a transmembrane domain comprising a human CD8 polypeptide that comprises an amino acid sequence of amino acids 137 to 209 of SEQ ID NO:7.
  • the CD8 polypeptide has an amino acid sequence that is at least about 85 %, about 90 %, about 95 %, about 96 %, about 97 %, about 98 %, about 99 % or about 100 % identical to the sequence having a NCBI Reference No: AAA92533.1, SEQ ID NO: 8, or fragments thereof, and/or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions.
  • the CD8 polypeptide can have an amino acid sequence that is a consecutive portion of SEQ ID NO: 8 which is at least about 20, or at least about 30, or at least about 40, or at least about 50, or at least about 60, or at least about 70, or at least about 100, or at least about 200, and up to 247 amino acids in length.
  • the CD8 polypeptide comprises or has an amino acid sequence of amino acids I to 247, 1 to 50, 50 to 100, 100 to 150, 150 to 200, 151 to 219, or 200 to 247 of SEQ ID NO: 8.
  • the CAR comprises a transmembrane domain comprising a murine CD8 polypeptide that comprises an amino acid sequence of amino acids 151 to 219 of SEQ ID NO:8.
  • the CD8 polypeptide comprises or has the amino acid sequence set forth in SEQ ID NO:9.
  • the transmembrane domain of a CAR comprises a CD28 polypeptide.
  • the CD28 polypeptide can have an amino acid sequence that is at least about 85 %, about 90 %, about 95 %, about 96 %, about 97 %, about 98 %, about 99 % or 100 % identical to the sequence having a NCBI Reference No: P10747 or NP_006130 (SEQ ID No: 10), or fragments thereof, and/or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions.
  • the CD28 polypeptide can have an amino acid sequence that is a consecutive portion of SEQ ID NO: 10 which is at least 20, or at least 30, or at least 40, or at least 50, and up to 220 amino acids in length.
  • the CD28 polypeptide has an amino acid sequence of amino acids 1 to 220, 1 to 50, 50 to 100, 100 to 150, 114 to 220, 150 to 200, or 200 to 220 of SEQ ID NO: 10.
  • the CD28 polypeptide comprised in the transmembrane domain of a presently disclosed CAR has an amino acid sequence of amino acids 153 to 179 of SEQ ID NO: 10.
  • the transmembrane domain of a CAR comprises a NKG2D polypeptide.
  • the NKG2D polypeptide can have an amino acid sequence that is at least about 85 %, about 90 %, about 95 %, about 96 %, about 97 %, about 98 %, about 99 % or 100 % identical to the sequence having a NCBI Reference No: NP_031386.2 (SEQ ID NO:66), or fragments thereof, and/or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions.
  • the NKG2D polypeptide can have an amino acid sequence that is a consecutive portion of SEQ ID NO:66 which is at least 20, or at least 30, or at least 40, or at least 50, and up to 216 amino acids in length.
  • the NKG2D polypeptide comprises the transmembrane domain of SEQ ID NO: 66 (e.g., amino acids 52-72).
  • the transmembrane domain of a CAR comprises a CD3zeta (CD3Q polypeptide.
  • the CD3zeta polypeptide can have an amino acid sequence that is at least about 85 %, about 90 %, about 95 %, about 96 %, about 97 %, about 98 %, about 99 % or 100 % identical to the sequence of the transmembrane domain of CD3zeta set forth in NCBI Reference No: NP_932170 (SEQ ID NO: 11), SEQ ID NO: 12, or SEQ ID NO: 13, and/or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions.
  • the transmembrane domain of a CAR comprises a CD4 polypeptide.
  • the CD4 polypeptide can have an amino acid sequence that is at least about 85 %, about 90 %, about 95 %, about 96 %, about 97 %, about 98 %, about 99 % or 100 % identical to the sequence of the transmembrane domain of CD4 set forth in NCBI Reference No: NP_000607.1, NP_001181943.1 or NP_001181946.1, (each incorporated herein by reference) and/or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions.
  • the transmembrane domain of a CAR comprises a 4- IBB polypeptide.
  • the 4- IBB polypeptide can have an amino acid sequence that is at least about 85 %, about 90 %, about 95 %, about 96 %, about 97 %, about 98 %, about 99 % or 100 % identical to the sequence of the transmembrane domain of 4- IBB set forth in NCBI Reference No: P41273 (incorporated herein by reference) or NP_001552.2 (SEQ ID NO: 18), and/or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions.
  • the transmembrane domain of a CAR comprises an OX40 polypeptide.
  • the OX40 polypeptide can have an amino acid sequence that is at least about 85 %, about 90 %, about 95 %, about 96 %, about 97 %, about 98 %, about 99 % or 100 % identical to the sequence of the transmembrane domain of OX40 set forth in NCBI Reference No: NP_OO3318.1 (incorporated herein by reference), and/or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions.
  • the transmembrane domain of a CAR comprises an ICOS polypeptide.
  • the ICOS polypeptide can have an amino acid sequence that is at least about 85 %, about 90 %, about 95 %, about 96 %, about 97 %, about 98 %, about 99 % or 100 % identical to the sequence of the transmembrane domain of ICOS set forth in NCBI Reference No: NP_036224.1 (incorporated herein by reference), and/or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions.
  • a CAR can also comprise a spacer region that links the extracellular antigen-binding domain to the transmembrane domain.
  • the spacer region can be flexible enough to allow the antigen binding domain to orient in different directions to facilitate antigen recognition.
  • the spacer region can be the hinge region from IgGl (GenPept Ref No.: P01857.1, incorporated herein by reference), or the CH2CH3 region of an immunoglobulin (e.g., IgG4 (GenPept Ref No.: P01861.1, incorporated herein by reference), a portion of a CD3 polypeptide, a portion of a CD28 polypeptide (e.g., a portion of SEQ ID NO: 10), a portion of a CD8 polypeptide (e.g., a portion of SEQ ID NO:7, or a portion of SEQ ID NO: 8), a variation of any of the foregoing which is at least about 80 %, at least about 85 %, at least about 90 %, or at least about 95 % identical thereto, or a synthetic spacer sequence.
  • intracellular signaling domain refers to the intracellular domain of a CAR which comprises one or more cytoplasmic or intracellular signaling domains, or portions of cytoplasmic of intracellular signaling domains, of cell surface or intracellular molecules such as activating or stimulatory molecules, co- stimulatory molecules, co-stimulatory ligands, and signal transduction molecules.
  • activating or stimulatory molecules refer to cell surface molecules such as antigen receptors or phagocytosis receptors or cytokine receptors or chemokine receptors, which can activate or stimulate a cell (e.g., a cell of the myeloid lineage, e.g., a monocyte and/or macrophage).
  • co-stimulatory molecules refer to cell surface molecules other than antigen receptors or their ligands that are required for an efficient response of immune cells to antigen, providing optimal cellular activation or suppression.
  • a “co-stimulatory ligand” refers to a protein expressed on the cell surface that upon binding to its co-stimulatory receptor produces a co-stimulatory response, i.e., an intracellular response that effects the stimulation provided when an antigen binds to its receptor.
  • signal transduction molecules refer to intracellular proteins, such as signaling adaptors, that participate in the propagation of a signaling cascade in response to binding of extracellular or intracellular receptors to their cognate ligands.
  • an intracellular signaling domain of the CAR can comprise one or more intracellular signaling domains derived from stimulatory molecules, co-stimulatory molecules, co-stimulatory ligands or signal transduction molecules including but not limited to CD3zeta (CD3Q, CD3delta, CD3epsilon, CD3gamma, CD4, CD8A, CD8B, CD2, CD7, LIGHT, CD27, CD28, 4-1BB (CD137), CD226 (DNAM1), B24 (CD244), ICOS (CD278), CTLA-4, GITR, OX40 (CD134), LAT, PD-1, TIM3, TIGIT, PD-L1, PD-L2, OX40L, 4-1BBL, ICOSLG, CD30L, CD30, CD36, CD68, CD40, CD70, CD80, CD83, CD86, CD163, CD204 (MSR1), CD206 (MRC1), CD209 (DC-SIGN), AGER
  • chemokine receptors including but not limited to CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CCR11, CXCR1, CXCR2, CXCR3, CXCR3B, CXCR4, CXCR5, CXCR6, CXCR7, XCR1, or CX3CR1), or any combination thereof.
  • an intracellular signaling domain of the CAR can comprise a CD3zeta (CD3Q polypeptide.
  • CD3( ⁇ comprises 3 immunoreceptor tyrosinebased activation motifs (IT AMs) and transmits an activation signal to the cell (e.g., a cell of the myeloid lineage, e.g., a monocyte and/or macrophage) after antigen is bound.
  • I AMs immunoreceptor tyrosinebased activation motifs
  • the CD3zeta polypeptide has an amino acid sequence that is at least about 85 %, about 90 %, about 95 %, about 96 %, about 97 %, about 98 %, about 99 % or about 100 % identical to the sequence having a NCBI Reference No: NP_932170 (SEQ ID NO: 11), or fragments thereof, and/or can optionally comprise up to one or up to two or up to three conservative amino acid substitutions.
  • the CD3( ⁇ polypeptide comprises or has an amino acid sequence that is a consecutive portion of SEQ ID NO: 11, which is at least 20, or at least 30, or at least 40, or at least 50, and up to 164 amino acids in length.
  • the CD3zeta polypeptide comprises or has an amino acid sequence of amino acids 1 to 164, 1 to 50, 50 to 100, 100 to 150, or 150 to 164 of SEQ ID NO: 11.
  • the CD3 ⁇ polypeptide comprises or has an amino acid sequence of amino acids 52 to 164 of SEQ ID NO: 11.
  • the CD3zeta polypeptide has an amino acid sequence that is at least about 85 %, about 90 %, about 95 %, about 96 %, about 97 %, about 98 %, about 99 % or about 100 % identical to the sequence having a NCBI Reference No: NP_001106864.2 (SEQ ID NO: 12), or fragments thereof, and/or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions.
  • the CD3zeta polypeptide can have an amino acid sequence that is a consecutive portion of SEQ ID NO: 12, which is at least about 20, or at least about 30, or at least about 40, or at least about 50, or at least about 90, or at least about 100, and up to 188 amino acids in length.
  • the CD3 ⁇ polypeptide comprises or has an amino acid sequence of amino acids 1 to 164, 1 to 50, 50 to 100, 52 to 142, 100 to 150, or 150 to 188 of SEQ ID NO: 12.
  • the CD3zeta polypeptide comprises or has an amino acid sequence of amino acids 52 to 142 of SEQ ID NO: 12.
  • the CD3zeta polypeptide comprises or has the amino acid sequence set forth in SEQ ID NO: 13.
  • the intracellular signaling domain of the CAR can comprise a CD28 polypeptide.
  • the CD28 polypeptide can have an amino acid sequence that is at least about 85 %, about 90 %, about 95 %, about 96 %, about 97 %, about 98 %, about 99 % or 100 % identical to the sequence having a NCBI Reference No: P10747 (incorporated herein by reference) or NP_006130 (SEQ ID NO: 10), or fragments thereof, and/or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions.
  • the CD28 polypeptide has an amino acid sequence that is a consecutive portion of SEQ ID NO: 10 which is at least 20, or at least 30, or at least 40, or at least 50, and up to 220 amino acids in length.
  • the CD28 polypeptide has an amino acid sequence of amino acids 1 to 220, 1 to 50, 50 to 100, 100 to 150, 114 to 220, 150 to 200, or 200 to 220 of SEQ ID NO: 10.
  • the intracellular signaling domain of the CAR comprises a signaling region that comprises a CD28 polypeptide having an amino acid sequence of amino acids 180 to 220 of SEQ ID NO: 10.
  • the CD28 polypeptide has an amino acid sequence that is at least about 85 %, about 90 %, about 95 %, about 96 %, about 97 %, about 98 %, about 99 % or about 100 % identical to the sequence having a NCBI Reference No: NP_031668.3 (SEQ ID NO: 14), or fragments thereof, and/or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions.
  • the CD28 polypeptide has an amino acid sequence that is a consecutive portion of SEQ ID NO: 14 which is at least about 20, or at least about 30, or at least about 40, or at least about 50, and up to 218 amino acids in length.
  • the CD28 polypeptide has an amino acid sequence of amino acids 1 to 218, 1 to 50, 50 to 100, 100 to 150, 114 to 220, 150 to 200, 178 to 218, or 200 to 220 of SEQ ID NO: 14.
  • the signaling region of a presently disclosed CAR comprises a CD28 polypeptide that comprises or has the amino acids 178 to 218 of SEQ ID NO: 14.
  • the intracellular signaling domain of the CAR comprises an intracellular signaling region that comprises two intracellular signaling molecules: CD28 (SEQ ID NO: 10; SEQ ID NO: 14) and 4-1BB or CD28 and OX40.
  • the 4- IBB can act as a tumor necrosis factor (TNF) ligand and have stimulatory activity.
  • the 4- IBB polypeptide can have an amino acid sequence that is at least about 85 %, about 90 %, about 95 %, about 96 %, about 97 %, about 98 %, about 99 % or about 100 % identical to the sequence set forth in NCBI Reference No: P41273 (incorporated herein by reference) or NP_001552.2 (SEQ ID NO: 18) or fragments thereof, and/or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions.
  • an OX40 polypeptide can have an amino acid sequence that is at least about 85 %, about 90 %, about 95 %, about 96 %, about 97 %, about 98 %, about 99 % or about 100 % identical to the sequence having a NCBI Reference No: P43489 (incorporated herein by reference) or NP_OO3318.1 (SEQ ID NO: 19), or fragments thereof, and/or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions.
  • an ICOS polypeptide can have an amino acid sequence that is at least about 85 %, about 90 %, about 95 %, about 96 %, about 97 %, about 98 %, about 99 % or about 100 % identical to the sequence having a NCBI Reference No: NP_036224.1 (SEQ ID NO:20) or fragments thereof, and/or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions.
  • a DAP- 12 polypeptide can have an amino acid sequence of a co-stimulatory region that is at least about 85 %, about 90 %, about 95 %, about 96 %, about 97 %, about 98 %, about 99 % or about 100 % identical to the sequence having a NCBI Reference No: NP_003323.1, NP_001166986.1, NP_001166985.1 or NP_937758.1 (each incorporated herein by reference) or fragments thereof, and/or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions.
  • a 2B4 polypeptide can have an amino acid sequence of a co-stimulatory region that is at least about 85 %, about 90 %, about 95 %, about 96 %, about 97 %, about 98 %, about 99 % or about 100 % identical to the sequence having a NCBI Reference No: NP_057466.1, NP_001160135.1 or NP_001160136.1 (each incorporated herein by reference) or fragments thereof, and/or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions.
  • the monocyte/macrophage cell product can be genetically modified to express non-CAR genetic constructs such as a receptor (such as a TCR, chimeric receptor, or other receptor), immune cell engager, antibody, nanobody, transcription factor, cytokine, and/or chemokine.
  • a receptor such as a TCR, chimeric receptor, or other receptor
  • immune cell engager antibody, nanobody, transcription factor, cytokine, and/or chemokine.
  • chimeric receptor refers to a cell surface, synthetic molecule comprising an extracellular domain, a transmembrane domain, and an intracellular signaling domain or domains from two or more molecules.
  • the term “immune cell engager” refers to a synthetic molecule comprising two or more binding domains designed to create an artificial immune synapse between tumor cells and immune cells for the purpose of redirecting immune effector cell function.
  • the genetic modification is expression of a non-modified endogenous receptor.
  • the genetic modification is expression of a T cell receptor (TCR).
  • TCR T cell receptor
  • a TCR consists of one alpha and one beta chain, each containing a variable domain.
  • heterodimerization of the alpha and beta chains creates a unique receptor conformation that binds to HLA molecules displaying tumor-associated or virus-associated antigens on the surface of an antigen-presenting immune cell.
  • the TCR binds specifically to antigens associated with tumors including but not limited to melanoma, synovial sarcoma, lung, breast, ovarian, gastric, or pancreatic.
  • the TCR binds specifically to antigens associated with infectious diseases including but not limited to hepatitis B virus (HBV) or human immunodeficiency virus (HIV).
  • the genetic modification is expression of a chimeric receptor.
  • a chimeric receptor includes an extracellular domain, a transmembrane, and one or more intracellular signaling domains.
  • a chimeric receptor may include a linker between the extracellular domain and transmembrane domain, between the transmembrane domain and intracellular signaling domain, and/or between multiple intracellular signaling domains.
  • the endogenous receptor or the chimeric receptor can comprise all or a portion of molecules such as, but not limited to cytokine receptors, chemokine receptors, growth factor receptors, stimulatory molecules, costimulatory molecules, or costimulatory ligands.
  • the linker may be the (G4S)3 linker (SEQ ID NO:28).
  • the genetic modification is expression of an immune cell engager.
  • the immune cell engager includes one binding domain recognizing a tumor-associated antigen and one binding domain recognizing an immune cell surface protein.
  • the immune cell engager includes more than one binding domain recognizing a tumor-associated antigen and one binding domain recognizing an immune cell surface protein.
  • the immune cell engager includes one binding domain recognizing a tumor-associated antigen and more than one binding domain recognizing immune cell surface proteins.
  • the binding domains of the immune cell engager are comprised of scFv domains.
  • the immune cell engager binds specifically to tumor-associated antigens including but not limited to HER2, EpCAM, PSMA, PD-L1, CEA, EGFR, or CD33. In certain embodiments, the immune cell engager binds specifically to proteins on T cells including but not limited to CD3, CD28, and/or 4- IBB. In certain embodiments, the immune cell engager binds specifically to proteins on NK cells including but not limited to CD56, CD16, and/or NKG2D. In certain embodiments, the immune cell engager binds specifically to proteins on monocytes or macrophages including but not limited to CD64, CD40, CD80, and/or CD86.
  • the genetic modification is expression of an antibody or nanobody.
  • the genetic modification is a monoclonal antibody that comprises four polypeptide chains including two identical heavy and two light chains covalently bonded by mainly disulfide interactions into a Y-shaped structure of -150 kDa.
  • the monoclonal antibody comprises antigen binding fragments consisting of the variable regions of the heavy and light chains, herein referred to as Fv, responsible for specific binding to an antigen.
  • the Fv region is specific for a tumor-specific antigen or tumor-associated antigen including but not limited to CD20, EGFR, HER2, PD-1, TNF-alpha, IL-lbeta, IL-1RI, IL-4R, IL-6, IL-17A, or IL-23.
  • the genetic modification is a nanobody comprised of the single variable domain of - 15 kDa derived from a heavy-chain-only antibody, responsible for specific binding to an antigen, herein referred to as VHH.
  • the VHH region is specific for a tumor- specific or tumor-associated antigen including but not limited to EGFR, EGF, HER2, PD-1, CAIX, death receptor 5 (DR5), c-Met, mesothelin, or CD33.
  • the genetic modification is expression of a transcription factor or transcription factor subunit.
  • the transcription factor or transcription factor subunit modulates the function of the genetically engineered cell expressing the transcription factor.
  • the transcription factor or transcription factor subunit can derive from, for example, the following families of transcription factors: C/EBP family, NF-kB family, STAT family, KLF family, PPAR family, AP-1 family, NF AT family, GATA family, CREB family or IRF family.
  • the genetic modification is expression of a cytokine. In certain embodiments, the genetic modification is expression of a chemokine. In certain embodiments, the cytokine or chemokine modulates the surrounding microenvironment. In certain embodiments, cytokine or chemokine modulates the function or activities of immune cells such as T cells, NK cells, B cells, monocytes, macrophages, or dendritic cells, including the genetically engineered cell secreting the cytokine or chemokine.
  • immune cells such as T cells, NK cells, B cells, monocytes, macrophages, or dendritic cells
  • the cytokine or chemokine can include but is not limited to: interleukins (such as IL-lalpha, IL-lbeta, IE-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-17, IL-18, IL-21, IL-22), IFN-alpha, IFN-beta, IFN-gamma, IFN-lambda, TGFbeta, TNF-alpha, TNF-beta, GM-CSF, M-CSF, CCL1, CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL11, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, C
  • the genetic modification is a receptor (such as a CAR, TCR, chimeric receptor, or other receptor), an immune cell engager, an antibody, a nanobody, a transcription factor, a cytokine, a chemokine, or any combination thereof.
  • a receptor such as a CAR, TCR, chimeric receptor, or other receptor
  • an immune cell engager such as an antibody, a nanobody, a transcription factor, a cytokine, a chemokine, or any combination thereof.
  • multiple genes are separated by a self-cleaving peptide, including but not limited to T2A or P2A.
  • the genetic modification further includes a gene(s) encoding an additional gene product, such as a transfection marker or a suicide gene such as truncated EGFR (tEGFR) (see WO2011/056894; SEQ ID NO:67) or iCasp9 (W02013/040371), the sequences of which are incorporated by reference herein.
  • an additional gene product such as a transfection marker or a suicide gene such as truncated EGFR (tEGFR) (see WO2011/056894; SEQ ID NO:67) or iCasp9 (W02013/040371), the sequences of which are incorporated by reference herein.
  • a CAR comprises an extracellular antigen-binding domain that binds to CD 19, a transmembrane domain comprising a CD28 polypeptide, and an intracellular signaling domain comprising a CD3zeta polypeptide and a co-stimulatory signaling region comprising a CD28 polypeptide.
  • the CAR is designated 1928z.
  • 1928z is a protein having at least about 85 %, about 90 %, about 95 %, about 96 %, about 97 %, about 98 %, about 99 % or about 100 % identical to the amino acid sequence set forth in SEQ ID NO:21.
  • the protein sequence includes a CD8 leader sequence at amino acids 1 to 18 and is able to bind human CD 19.
  • a CAR having an extracellular antigen-binding domain that binds to CD 19, a hinge spacer from human IgG4, a transmembrane domain comprising a CD28 transmembrane domain, a 4- IBB co-stimulatory signaling region, and a CD3 ⁇ intracellular signaling domain is provided.
  • the CAR is designated 1928zl.
  • 1928zl is a protein having at least about 85 %, about 90 %, about 95 %, about 96 %, about 97 %, about 98 %, about 99 % or about 100 % identical to the amino acid sequence set forth in SEQ ID NO:22.
  • a CAR comprises an extracellular antigen-binding domain that binds to MUC16, a transmembrane domain comprising a CD28 polypeptide, and an intracellular signaling domain comprising a CD3zeta polypeptide and a co-stimulatory signaling region comprising a CD28 polypeptide.
  • the CAR is designated 4H1128z.
  • 4H1128z is a protein having at least about 85 %, about 90 %, about 95 %, about 96 %, about 97 %, about 98 %, about 99 % or about 100 % identical to the amino acid sequence set forth in SEQ ID NO:23.
  • the protein includes a CD8 leader sequence at amino acids 1 to 18 and binds to the MUC-16 ectodomain.
  • a CAR comprises an extracellular antigen-binding domain that binds to CD 19, a transmembrane domain comprising a CD8 polypeptide, and an intracellular signaling domain comprising a CD3zeta polypeptide and a co-stimulatory signaling region comprising a 4- IBB polypeptide.
  • the CAR is 19BBz.
  • An exemplary protein sequence of the 19BBz polypeptide is set forth in SEQ ID NO:24.
  • a CAR having an extracellular antigen-binding domain that binds to CD 19, a hinge spacer from human IgG4, a transmembrane domain comprising a CD28 transmembrane domain, a 4- IBB co-stimulatory signaling region, and a CD3zeta intracellular signaling domain is provided.
  • An exemplary CD 19 CAR protein sequence, that also includes a truncated EGFR (tEGFR) polypeptide attached to the carboxy terminal portion of the CAR by a self-cleaving 2A peptide, is set forth in SEQ ID NO:61.
  • a CAR having an extracellular antigen-binding domain that binds to CD 19, a hinge spacer from human IgG4, a transmembrane domain of a CD28 transmembrane domain, a 4- IBB co- stimulatory signaling region, and a CD3( ⁇ intracellular signaling domain is provided.
  • An exemplary CD 19 CAR protein sequence, that also includes a human IL- 15 attached to the carboxy terminal portion of the CAR by a self-cleaving 2A peptide, is set forth in SEQ ID NO:62.
  • SEQ ID NO:69 The corresponding nucleic acid sequence that can encode the construct in SEQ ID NO:62 is set forth in SEQ ID NO:69.
  • a CAR having an extracellular antigen-binding domain that binds to CD 19, a hinge spacer from human IgG4, a transmembrane domain of a CD28 transmembrane domain, a 2B4 co- stimulatory signaling region, and a CD3zeta intracellular signaling domain is provided.
  • An exemplary CD 19 CAR protein sequence, that also includes a human IL- 15 attached to the carboxy terminal portion of the CAR by a selfcleaving 2A peptide, is set forth in SEQ ID NO:70.
  • SEQ ID NO:70 The corresponding nucleic acid sequence encoding the SEQ ID NO:70 construct is set forth in SEQ ID NO:71.
  • a CAR having an extracellular antigen-binding domain that binds to CD 19, a hinge spacer from human IgG4, a transmembrane domain of a NKG2D transmembrane domain, a 4- IBB co-stimulatory signaling region, and a CD3zeta intracellular signaling domain is provided.
  • Use of the NKG2Ds transmembrane domain has been used previously (Xu el al.. J. Hematol. Oncol. 12:49, 2019) although some suggest that because NKGD2 is a type II membrane protein it should be inserted in the opposite orientation.
  • An exemplary CD 19 CAR protein sequence, that also includes a human IL- 15 attached to the carboxy terminal portion of the CAR by a self-cleaving 2A peptide, is set forth in SEQ ID NO:72.
  • SEQ ID NO:72 The corresponding nucleic acid sequence encoding the SEQ ID NO:72 construct is set forth in SEQ ID NO:73.
  • a CAR having an extracellular antigen-binding domain that binds to CD 19, a hinge spacer from human IgG4, a transmembrane domain of a NKG2D transmembrane domain, a 2B4 co- stimulatory signaling region, and a CD3( ⁇ intracellular signaling domain is provided.
  • An exemplary CD 19 CAR protein sequence, that also includes a human IL- 15 attached to the carboxy terminal portion of the CAR by a self-cleaving 2A peptide, is set forth in SEQ ID NO:74.
  • SEQ ID NO:74 The corresponding nucleic acid sequence encoding the SEQ ID NO:74 construct is set forth in SEQ ID NO:75.
  • a receptor such as a CAR, TCR, chimeric receptor, or other receptor
  • an immune engager an antibody, a nanobody, a transcription factor, a cytokine, and/or a chemokine
  • a nucleic acid comprising an inducible promoter, for expressing nucleic acid sequences in human cells.
  • the inducible promoter is responsive to factors in the tumor microenvironment. In certain embodiments, the inducible promoter is responsive to exogenously administered molecules.
  • a receptor such as a CAR, TCR, chimeric receptor, or other receptor
  • an immune cell engager an antibody, a nanobody, a transcription factor, a cytokine, and/or a chemokine
  • a nucleic acid comprising a constitutive promoter, such as promoters for ubiquitin C (UbiC), PGK, EF-1 alpha, MND (a synthetic viral promoter that contains the U3 region of a modified Moloney murine leukemia virus long terminal repeat with myeloproliferative sarcoma virus enhancer), or Chicken beta actin.
  • a constitutive promoter such as promoters for ubiquitin C (UbiC), PGK, EF-1 alpha, MND (a synthetic viral promoter that contains the U3 region of a modified Moloney murine leukemia virus long terminal repeat with myeloproliferative sarcoma virus enhancer), or Chicken beta actin.
  • Methods of preparing genetically modified monocytes/macrophage compositions and/or preparations for immunotherapy comprise introducing into the CDl lb + HLA-DR + (mainly monocyte/macrophage) cells a polynucleotide(s) encoding a receptor (such as a CAR, TCR, chimeric receptor, or other receptor), an immune cell engager, an antibody, a nanobody, a transcription factor, a cytokine, and/or a chemokine) into the cells.
  • a receptor such as a CAR, TCR, chimeric receptor, or other receptor
  • the polynucleotide(s) encoding the desired molecule can be introduced into the HSPCs, before, during, or after expansion, or into HSPCs or monocyte/macrophage compositions and/or preparations before, during, or after differentiation.
  • Some embodiments relate to a method of engineering a monocyte/macrophage composition and/or preparation by transforming, transducing, or transfecting an HSPC or monocyte/macrophage composition and/or preparation with at least one polynucleotide encoding a receptor (such as a CAR, a TCR, a chimeric receptor, or other receptor), an immune cell engager, an antibody, a nanobody, a transcription factor, a cytokine, and/or a chemokine, and expressing the polynucleotide in the cell.
  • a receptor such as a CAR, a TCR, a chimeric receptor, or other receptor
  • Desired polynucleotides encoding genes can be introduced into HSPCs or other cells by any method known in the art, including transfection, electroporation, microinjection, lipofection, calcium phosphate mediated transfection, infection with a viral or bacteriophage vector containing the gene sequences, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, using CRISPR or other rare-cutting endonuclease (e.g., TALE-nuclease or Cas9 endonuclease), and the like. Numerous techniques are known in the art for the introduction of foreign genes into cells (see e.g., Loeffler and Behr, Meth.
  • the technique should provide for the stable transfer of the gene to the cell, so that the gene is expressible by the cell and preferably heritable and expressible by its cell progeny.
  • the method of transfer includes the transfer of a selectable marker or tag sequence to the cells. The cells are then placed under selection to isolate those cells that have taken up and are expressing the transferred gene.
  • the polynucleotide(s) or genes are included in lentiviral vectors in view of being stably expressed in the cells.
  • a receptor such as a CAR, a TCR, a chimeric receptor, or other receptor
  • an immune cell engager an antibody, a nanobody, a transcription factor, a cytokine, and/or a chemokine is introduced into HSPCs during the expansion phase and prior to differentiation, to form a receptor- (such as a CAR, TCR, chimeric receptor, or other receptor), an immune cell engager-, an antibody-, a nanobody-, a transcription factor-, a cytokine-, and/or a chemokine-expressing HSPCs and/or a receptor- (such as a CAR, TCR, chimeric receptor, or other receptor), an immune cell engager-, an antibody-, a nanobody-, a transcription factor-, a cytokine-, and/or a chemokine-expressing monocyte/macrophage.
  • a receptor- such as a CAR, TCR, chimeric receptor, or other receptor
  • the receptor- (such as a CAR, TCR, chimeric receptor, or other receptor), immune cell engager-, antibody-, nanobody-, transcription factor-, cytokine-, and/or chemokine is introduced into HSPCs during the expansion phase and prior to differentiation, to form receptor- (such as a CAR, TCR, chimeric receptor, or other receptor), immune cell engager-, antibody-, nanobody-, transcription factor-, cytokine-, and/or chemokine-expressing HSPCs and/or monocyte/macrophage.
  • receptor- such as a CAR, TCR, chimeric receptor, or other receptor
  • a receptor- such as a CAR, TCR, chimeric receptor, or other receptor
  • an immune cell engager-, an antibody-, a nanobody-, a transcription factor-, a cytokine-, and/or a chemokine is introduced into CDl lb + HLA-DR + monocyte/macrophage after expansion and during the differentiation phase, to form a receptor- (such as a CAR, a TCR, a chimeric receptor, or other receptor), an immune cell engager-, an antibody-, a nanobody-, a transcription factor-, a cytokine-, and/or a chemokine-expressing HSPCs and/or a receptor- (such as a CAR, a TCR, a chimeric receptor, or other receptor), an immune cell engager-, an antibody-, a nanobody-, a transcription factor-, a cytokine-, and/or a chemokine-expressing monocyte/macrophage
  • the receptor (such as a CAR, TCR, chimeric receptor, or other receptor), immune cell engager, antibody, nanobody, transcription factor, cytokine, and/or chemokine is introduced into CDl lb + HLA-DR + monocyte/macrophage after expansion and during the differentiation phase, to form a receptor- (such as a CAR, TCR, chimeric receptor, or other receptor), an immune cell engager-, an antibody-, a nanobody-, a transcription factor-, a cytokine-, and/or a chemokine-HSPCs and/or a receptor- (such as a CAR, TCR, chimeric receptor, or other receptor), an immune cell engager-, an antibody-, a nanobody-, a transcription factor-, a cytokine-, and/or a chemokine-expressing- monocyte/macrophage cells.
  • a receptor- such as a CAR, TCR, chimeric receptor, or other receptor
  • the receptor (such as a CAR, TCR, chimeric receptor, or other receptor), immune cell engager, antibody, nanobody, transcription factor, cytokine, and/or chemokine introduction, cell expansion, and cell differentiation mentioned above are each carried out as described above and below.
  • a receptor such as a CAR, TCR, chimeric receptor, or other receptor
  • an immune cell engager an antibody, a nanobody, a transcription factor, a cytokine, and/or a chemokine
  • a receptor such as a CAR, TCR, chimeric receptor, or other receptor
  • an immune cell engager an antibody, a nanobody, a transcription factor, a cytokine, and/or a chemokine
  • the plasmid vector can also contain a selection marker which provides for identification and/or selection of cells which received said vector.
  • Polypeptides such as a receptor (such as a CAR, a TCR, a chimeric receptor, or other receptor), an immune cell engager, an antibody, a nanobody, a transcription factor, a cytokine, and/or a chemokine, can be synthesized in situ in the cell as a result of the introduction of polynucleotides encoding the polypeptides into the cell.
  • a receptor such as a CAR, a TCR, a chimeric receptor, or other receptor
  • an immune cell engager an antibody
  • a nanobody such as a CAR, a TCR, a chimeric receptor, or other receptor
  • a transcription factor such as a cytokine, and/or a chemokine
  • Methods for viral mediated introduction of a polynucleotide construct into cells are known in the art. and include as non-limiting examples recombinant viral vectors (e.g., lentiviruses, retroviruses, adenoviruses, and AAVs).
  • recombinant viral vectors e.g., lentiviruses, retroviruses, adenoviruses, and AAVs.
  • An oncolytic virus can be used as a form of immunotherapy in which the virus selectively replicates within and lyses tumor cells, leading to activation of innate immune cells including, but not limited to, monocytes, macrophages, dendritic cells, or NK cells.
  • innate immune cells including, but not limited to, monocytes, macrophages, dendritic cells, or NK cells.
  • Viral oncolysis of tumor cells results in dispersal of viral progeny and antigen release.
  • An innate function of macrophages is phagocytic activity to clear virus particles and virally infected cells.
  • Macrophage phagocytic activity can result in immune activation including, but not limited to, release of cytokines, a shift in tumor-resident macrophages from a tumor- supportive phenotype towards an anti-tumor pro-inflammatory phenotype, sustained inflammation, recruitment of immune cells to infiltrate the suppressive tumor microenvironment (TME), or macrophage-mediated antigen cross-presentation to T cells to initiate a cascade of adaptive immune responses against the tumor cells.
  • an oncolytic virus comprises modified herpes simplex virus, adenovirus, vaccinia virus, myxoma virus, measles virus, Coxsackievirus, parvovirus, or human intestinal cytopathic orphan virus.
  • oncolytic virus is delivered through systemic administration, intratumoral injection, or via carrier cells pre-loaded with virus ex vivo prior to administration.
  • carrier cells comprise NK cells, monocytes, dendritic cells, and/or macrophages.
  • carrier cells comprise the monocyte/macrophage product.
  • a monocyte/macrophage composition and/or preparation, with or without genetic engineering, can be divided and frozen in one or more bags (or units).
  • from about 50 to about 500 million total cells are frozen in a single bag (or unit).
  • from about 100 to about 500 million cells are frozen in a single bag (or unit).
  • about 50, 100, 200, 300, 400 or 400 million cells are frozen in a single bag (or unit).
  • a single bag (or unit) contains about 50 million to about 2 billion viable cells per dose.
  • a single bag (or unit) contains about 100 million, about 200 million, about 300 million, about 400 million, about 500 million, about 600 million, about 750 million, about 1 billion, about 1.5 billion or about 2 billion viable cells. In some embodiments, a single bag contains about 50 million to about 2 billion viable CDl lb + HLA-DR + monocyte/macrophage per dose. In some embodiments, a single bag (or unit) contains about 100 million, about 200 million, about 300 million, about 400 million, about 500 million, about 600 million, about 750 million, about 1 billion, about 1.5 billion or about 2 billion viable CD1 lb + HLA-DR + monocyte/macrophage.
  • the monocyte/macrophage composition and/or preparation is frozen or cryopreserved.
  • the monocyte/macrophage composition and/or preparation is fresh, the cells have not been previously frozen prior to expansion or cryopreservation.
  • the terms “frozen/freezing” and “cryopreserved/cryopreserving/cryopreservation” are used interchangeably in the present application. Cryopreservation can be by any method known in the art that preserves cells in viable form. The freezing of cells is ordinarily destructive because on cooling, water within the cell freezes, leading to injury caused by osmotic effects on the cell membrane, cell dehydration, solute concentration, and ice crystal formation.
  • Cryoprotective agents which can be used include, but are not limited to, dimethyl sulfoxide (DMSO) (Lovelock and Bishop, Nature 183: 1394-1395, 1959; Ashwood-Smith, Nature 190: 1204-1205, 1961); glycerol, polyvinylpyrrolidine (Rinfret, Ann. N.Y. Acad. Sci. 85:576, 1960); polyethylene glycol (Sloviter and Ravdin, Nature 196:548, 1962); albumin, dextran, sucrose, ethylene glycol, i-erythritol, D-ribitol, D-mannitol (Rowe et al., Fed. Proc.
  • DMSO dimethyl sulfoxide
  • glycerol polyvinylpyrrolidine
  • polyethylene glycol Rositer and Ravdin, Nature 196:548, 1962
  • albumin dextran, sucrose, ethylene glycol, i-erythritol, D-ribitol, D
  • DMSO is used.
  • DMSO is used at a concentration which is nontoxic to cells.
  • DMSO comprises up to about 20 % of the composition, up to about 15 % of the composition, up to about 10 % of the composition, up to about 5 % of the composition, up to about 2 % of the composition, up to about 1 % of the composition, or up to about 0.5 % of the composition.
  • addition of plasma can augment the protective effect of DMSO.
  • addition of a human protein such as for example, human serum albumin (e.g., up to a concentration of about 2 to 10 %) can augment the protective effect of DMSO.
  • cells should be kept at 0 C until freezing, since DMSO concentrations of about 1 % can be toxic at temperatures above 4° C.
  • PBS containing 20 % DMSO and 8 % human serum albumin (HSA), or other suitable cell freezing media is used.
  • This mixture is then diluted 1: 1 with media so that the final concentration of DMSO and HSA are 10 % and 4 %, respectively.
  • the cells in this mixture are then frozen to -80 °C at a rate of 1 °C per minute and stored in the vapor phase of a liquid nitrogen storage tank.
  • a controlled slow cooling rate can be important.
  • Different cryoprotective agents Rapatz et al.
  • a programmable freezing apparatus allows the determination of optimal cooling rates and facilitates standard reproducible cooling.
  • a programmable controlled-rate freezer such as Cryomed or Planar permit tuning of the freezing regimen to the desired cooling rate curve.
  • the optimal rate is 1 °C to 3 °C per minute from 0 °C to -80 °C.
  • this cooling rate of 1 °C to 3 °C per minute from 0 °C to -80 °C can be used.
  • the container holding the cells must be stable at cryogenic temperatures and allow for rapid heat transfer for effective control of both freezing and thawing.
  • Sealed plastic vials e.g., Nunc, the Wheaton Cryule®
  • glass ampules can be used for multiple small amounts (1 to 2 mL) or larger amounts (e.g. , 5 to 30 mL), while larger volumes (20 to 200 mL) can be frozen in polyolefin bags (e.g., Del-Med) or ethylene vinyl acetate freezer bags (e.g., OriGen) held between metal plates for better heat transfer during cooling.
  • polyolefin bags e.g., Del-Med
  • ethylene vinyl acetate freezer bags e.g., OriGen
  • bags of bone marrow cells have been successfully frozen by placing them in -80 °C freezers which gives a cooling rate of approximately 3 °C/minute.
  • the methanol bath method of cooling can be used.
  • the methanol bath method is well-suited to routine cryopreservation of multiple small items on a large scale. The method does not require manual control of the freezing rate nor a recorder to monitor the rate.
  • DMSO-treated cells are pre-cooled on ice and transferred to a tray containing chilled methanol, which is placed in a mechanical refrigerator (e.g., Harris or Revco) at -80 °C. Thermocouple measurements of the methanol bath and the samples indicate the desired cooling rate of 1 °C to 3 °C per minute. After at least two hours, the specimens have reached a temperature of -80 °C and can be placed directly into liquid nitrogen (-196 °C) for permanent storage.
  • a mechanical refrigerator e.g., Harris or Revco
  • samples are cryogenically stored in liquid nitrogen (-196° C) or its vapor (between about -140 °C and -180 °C).
  • samples are cryogenically stored in liquid nitrogen vapor phase (e.g., at about -140 °C to -180 °C).
  • Suitable racking systems are commercially available and can be used for cataloguing, storage, and retrieval of individual specimens.
  • cry opreservation of viable cells or modifications thereof, are available and envisioned for use (e.g., cold metal-mirror techniques; Livesey and Linner, Nature 327:255, 1987; Linner et al., J. Histochem. Cytochem. 34(9): 1123-1135, 1986; see also U.S. Patent No. 4,199,022 by Senkan et al., U.S. Patent No. 3,753,357 by Schwartz, U.S. Patent No. 4,559,298 by Fahy).
  • Cryopreserved or frozen cells are preferably thawed quickly (e.g., in a water bath maintained at 37 °C to 41 °C), and chilled immediately upon thawing.
  • the vial containing the frozen cells can be immersed up to its neck in a warm water bath with gentle rotation to ensure mixing of the cell suspension as it thaws and to increase heat transfer from the warm water to the internal ice mass. As soon as the ice has completely melted, the vial is immediately placed in ice.
  • a cryopreserved monocyte/macrophage composition and/or preparation is thawed, and the full preparation, or a portion thereof, is infused into a human or animal patient in need thereof (e.g., having an infection, for example, a viral infection (e.g., HIV, HSV1 or 1, Hepatitis A, B, or C, Zika, SARS-CoV or SARS-CoV-2, and the like); or other infection as disclosed herein).
  • a viral infection e.g., HIV, HSV1 or 1, Hepatitis A, B, or C, Zika, SARS-CoV or SARS-CoV-2, and the like
  • Several procedures relating to processing of the thawed cells are available, known in the art, and can be employed if desirable.
  • the cryoprotective agent if toxic in humans, should be removed prior to therapeutic use of the thawed monocyte/macrophage composition and/or preparation. In an embodiment employing DMSO as the cryopreservative, it is preferable to omit this step to avoid cell loss. However, where removal of the cryoprotective agent is desired, the removal is preferably accomplished upon thawing.
  • removal of the cryoprotective agent can be by dilution to achieve the cryoprotective agent at an insignificant concentration. This can be accomplished by an addition of medium, followed by, if necessary, one or more cycles of centrifugation to pellet cells, removal of the supernatant, and resuspension of the cells.
  • intracellular DMSO in the thawed cells can be reduced to a level (less than 1 %) that will not adversely affect the recovered cells. This is preferably done slowly to minimize potentially damaging osmotic gradients that occur during DMSO removal.
  • cell count e.g., by use of a hemocytometer
  • viability testing e.g., by trypan blue exclusion; Kuchler, Biochemical Methods in Cell Culture and Virology, Dowden, Hutchinson & Ross, Stroudsburg, Pa., pp. 18-19, 1977; Methods in Medical Research, Eisen et al., eds., Vol. 10, Year Book Medical Publishers, Inc., Chicago, pp. 39-47, 1964
  • the percentage of viable antigen (e.g., CD1 lb + HLA-DR + ) positive cells can be determined by calculating the number of antigen positive cells that exclude 7-AAD (or other suitable dye excluded by viable cells) in an aliquot of the cells, divided by the total number of nucleated cells (TNC) (both viable and non-viable) in the aliquot of the cells. The number of viable antigen positive cells can then be determined by multiplying the percentage of viable antigen positive cells by the TNC.
  • 7-AAD or other suitable dye excluded by viable cells
  • the total number of nucleated cells Prior to cryopreservation and/or after thawing, the total number of nucleated cells, or in a specific embodiment, the total number of CDl lb + HLA-DR + cells, can be determined. For example, total nucleated cell count can be performed by using a hemocytometer and exclusion of trypan blue dye. Specimens that are of high cellularity can be diluted to a concentration range appropriate for manual counting. Final cell counts for products are corrected for any dilution factors.
  • Total nucleated cell count equals viable nucleated cells per mL x volume of product in milliliters (mL).
  • the number of CD1 lb + HLA-DR + positive cells in the sample can be determined, e.g., by use of flow cytometry using anti-CDl lb and anti-HLA-DR monoclonal antibodies conjugated to at least one fluorochrome.
  • the monocyte/macrophage compositions and/or preparations can be used in immunotherapy for the treatment of viral disorders, bacterial infections, solid tumors, hematopoietic malignancies, autoimmune disorders, and the like.
  • the monocyte/macrophage compositions and/or preparations are administered to inhibit inflammation, viral/bacterial proliferation in a subject (also referred to as a patient) in need thereof.
  • a method according to this aspect of the present invention is affected by administering a therapeutically effective amount of a monocyte/macrophage composition and/or preparation to the subject.
  • treating includes, but is not limited to, the administration of a monocyte/macrophage composition and/or preparation to reduce or delay the onset of the symptoms, complications, or biochemical indicia of a disease, alleviating the symptoms or arresting or inhibiting further development of the disease, condition, or disorder (e.g., cancer, metastatic cancer, metastatic solid tumors, viral or bacterial symptoms).
  • Treatment can be prophylactic, i.e., as an adjuvant (to prevent relapse or delay the onset of the disease, or to prevent the manifestation of clinical or subclinical symptoms thereof) or therapeutic suppression or alleviation of symptoms after the manifestation of the disease.
  • a monocyte/macrophage cell composition and/or preparation is administered in an amount effective to inhibit inflammation, inhibit viral and/or bacterial infection, or inhibit tumor proliferation.
  • the following example demonstrates the ex vivo expansion and differentiation of a monocyte and/or macrophage product from hematopoietic stem cells and/or hematopoietic progenitor cells (HSPCs).
  • the HSPCs in this example are CD34+ cells isolated from human cord blood and/or human placental blood.
  • the method comprises three distinct linear phases of ex vivo culture, where Phase 1 comprises Expansion, Phase 2 comprises Expansion and Monocyte Differentiation, and Phase 3 comprises Macrophage Differentiation.
  • CD34+ HSPCs enriched from 2 to 20 pooled unmatched cord blood units, were seeded into tissue culture treated plastic culture vessels lacking any additional coating at a density of 9,000 - 10,000 cells/cm 2 .
  • the ex vivo culture medium used in the Phase 1 culturing step comprises X- VIVOTM- 10 medium (Lonza) supplemented with stem cell factor (SCF; 50 ng/mL), thrombopoietin (TPO; 50 ng/mL), FMS-like tyrosine kinase 3 ligand (Flt3-L; 50 ng/mL), and interleukin 6 (IL-6; 50 ng/mL), and interleukin 3 (IL-3;
  • Cells were cultured for 14 to 21 days, with either a feed with Phase 1 medium every 3 to 4 days or a harvest and replating of the cells in new vessels at a density of 20,000 - 30,000 cells/cm 2 on Day 7 and Day 14 (for the 21-day expansion) with additions of Phase 1 medium to accommodate increasing cell densities.
  • Cell harvesting consisted of collection of non-adherent cells with the culture medium into a sterile conical tube.
  • Adherent cells were incubated for 20 to 30 minutes with phosphate buffered saline (PBS), dislodged from the tissue culture plastic by pipetting, and combined with the non-adherent cells. All collected cells were then washed with PBS by centrifugation.
  • PBS phosphate buffered saline
  • Cells collected at the end of Phase 1 of culture could be either passaged directly into Phase 2 Expansion and Monocyte Differentiation or cryopreserved for later use using CryoStor® CS10 (Biolife Solutions) which comprises 10 % DMSO as the cryopreservation medium.
  • CryoStor® CS10 Biolife Solutions
  • Phase 1 HSPC Expansion cells were harvested and washed with phosphate buffered saline (PBS) by centrifugation, and resuspended in Phase 2 culture medium, comprising of X- VIVOTM- 10 medium (Lonza) supplemented with monocyte colony stimulating factor (M-CSF; 50 ng/mL), Flt3-L (50 ng/mL), and IL-3 (10 ng/mL).
  • PBS phosphate buffered saline
  • M-CSF monocyte colony stimulating factor
  • Flt3-L 50 ng/mL
  • IL-3 10 ng/mL
  • Cells were seeded into tissue culture treated plastic culture vessels lacking any additional coating at a density of 20,000 - 30,000 cells/cm 2 .
  • Cells were cultured for 7 days with a feed with Phase 2 medium 3 to 4 days into the culture period.
  • Cell harvesting at the end of Phase 2 of culture consisted of collection of non-adherent cells with the culture medium into a sterile conical tube.
  • Adherent cells were incubated for 20-30 minutes with phosphate buffered saline (PBS), dislodged from the tissue culture plastic by pipetting, and combined with the non-adherent cells. All collected cells were then washed with PBS by centrifugation.
  • Cells collected at the end of Phase 2 of culture could be either passaged directly into Phase 3 Macrophage Differentiation culture or cryopreserved for later use using CryoStor® CS10 (Biolife Solutions) as the cryopreservation medium.
  • CryoStor® CS10 Biolife Solutions
  • Phase 2 Expansion and Monocyte Differentiation culture cells were harvested and washed with PBS by centrifugation, and resuspended in Phase 3 culture medium, comprising of RPMI 1640 (Gibco), supplemented with granulocyte-macrophage colony stimulating factor (GM-CSF; 10 ng/mL) and either 2.5 % human platelet lysate (HPL) or 10 % fetal bovine serum (FBS).
  • RPMI 1640 Gibco
  • GM-CSF granulocyte-macrophage colony stimulating factor
  • HPL human platelet lysate
  • FBS fetal bovine serum
  • Cells were seeded into tissue culture treated plastic culture vessels lacking any additional coating at a density of 130,000 - 230,000 cells/cm 2 .
  • Cells were cultured for 7 days with a feed with Phase 3 medium 3 to 4 days into the culture period.
  • Phase 3 culture non-adherent cells were collected with the culture medium into a sterile conical tube.
  • Adherent cells were incubated with TrypLETM Express Enzyme (Gibco) according to product instructions, followed by scraping of the tissue culture plastic surface, and the released adherent cells were combined with the collected non-adherent cells and washed with RPMI 1640 by centrifugation. Washed cells could either be used for in vitro or in vivo analyses or cryopreserved for later use using CryoStor® CS10 (Biolife Solutions) as the cryopreservation medium.
  • CryoStor® CS10 Biolife Solutions
  • Ex vivo expansion of cells during Phase 1, Phase 2, and Phase 3 of culture generates approximately 78,000 cells from each starting CD34+ cell seeded into culture, with greater than 80 % viability maintained throughout the culture process (see FIGS. 1A and IB).
  • Directed differentiation of the expanded cell population that occurs during Phase 2 and Phase 3 of culture results in 60 to 90 % of the cells being identified as monocytes or macrophages, generating approximately 54,000 monocytes and macrophages from each starting CD34+ cell seeded into culture (see FIGS. 2A and 2B).
  • Monocytes generated above express the cell surface proteins HLA-DR and CD 11b as well as the cell surface receptors CD14 and/or CD16.
  • the HLA-DR+ CDl lb+ monocyte population makes up 40 to 80 % of the total cell population (see FIGS. 2A and 2B and FIGS. 3 A and 3B).
  • an average of 4.1 % +/- 4.1 % of the total cells are CD14+ CD16', classified as Classical monocytes, an average of 66.7 % +/- 10.8 % of the total cells are CD14+ CD16+, classified as Intermediate monocytes, and an average of 8.4 % +/- 6.9 % of the total cells are CD14 low CD16+, classified as Non-Classical monocytes.
  • Macrophages differentiated from the monocytes generated through the current invention also express the cell surface proteins HLA-DR and CD1 lb as well as the cell surface receptors CD14 and/or CD16.
  • the HLA-DR+ CDl lb+ macrophage population makes up 60 - 90 % of the total cell population (see FIGS. 2A and 2B and FIGS. 3A-3C).
  • the total cell population at the end of the Macrophage Differentiation expresses CD 14 and CD 16 comparable to that observed at the end of the Expansion and Monocyte Differentiation (Phase 2 of culture) (7.2 % +/- 7.2 % CD14+ CD16-; 66.9 % +/- 11.5 % CD14+ CD16+; 5.1 % +/- 3.1 % CD14- CD16+).
  • the remaining cell population that is not HLA-DR+ CDl lb + is comprised of myeloid-derived cells, including dendritic cells.
  • myeloid-derived cells including dendritic cells.
  • GM-CSF granulocyte-macrophage colony stimulating factor
  • M-CSF macrophage colony stimulating factor
  • additional markers are expressed on these macrophages that are associated with either an Ml polarization (e.g., CD40, CD86) or an M2 polarization (e.g., CD163, CD206, CD209), respectively (see FIGS. 4A - 4D).
  • Both monocytes and macrophages generated through the current methods are capable of phagocytosis, as demonstrated by in vitro phagocytosis assays using the pH— sensitive pHrodoTM S. aureus BioparticlesTM that become fluorescent within the acidic environment of the cytoplasmic phagosome (see FIG. 5).
  • Both monocytes and macrophages can be cryopreserved, with high levels of cell recovery and viability post-thaw (see FIG. 6). Cryopreserved monocytes and macrophages also retain their capacity for phagocytosis following thaw (see FIG. 7). This demonstrated phagocytosis can include traditional macrophage targets like bacteria and other pathogens, dead cells and debris, and tumor cells.
  • these macrophages When these macrophages are activated by pathogens or tumor cells, or encounter dead cells and debris during wound healing, they can release cytokines associated with either Ml or M2 polarization, such as Tumor Necrosis Factor-alpha (TNFa) and IL- 12 by activated Ml macrophages or IL- 10 by immunomodulatory M2 macrophages, for example.
  • cytokines associated with either Ml or M2 polarization such as Tumor Necrosis Factor-alpha (TNFa) and IL- 12 by activated Ml macrophages or IL- 10 by immunomodulatory M2 macrophages, for example.
  • a CD 19 CAR-expressing monocytes and/or macrophages can be generated from hematopoietic stem cells and/or hematopoietic progenitor cells (HSPCs) that were engineered to express a CD 19 CAR construct using viral transduction.
  • the HSPCs in this example are CD34+ cells isolated from human cord blood and/or human placental blood.
  • the method comprises the three distinct linear phases of ex vivo culture as described in Example 1, modified to include viral transduction of HSPCs during the Phase 1 Expansion culture as described below.
  • CD34+ HSPCs were initiated into culture as described in Example 1. On day 2 of culture, the Phase 1 media overlying the cells was replaced with CD 19 CAR viral vector-conditioned media in a volume sufficient to completely submerge the cells. The tissue culture vessel containing the cells and vector-conditioned media was then centrifuged at 1400 x g for 1 hour at room temperature ( ⁇ 25 °C). Immediately following centrifugation, the tissue culture vessel was incubated for 2 hours in a humidified incubator set to 37°C and 5% CO2.
  • the transduction process was then repeated by replacing the spent vector-conditioned media with fresh vector-conditioned media as described above, and the tissue culture vessel containing the cells and fresh vector-conditioned media was centrifuged at 1400 x g for 1 hour at room temperature ( ⁇ 25 °C). Immediately following the second centrifugation, the tissue culture vessel was incubated for 1 hour in a humidified incubator set to 37°C and 5% CO2. Following this incubation, the spent vector-conditioned media was replaced with fresh Phase 1 media, and the tissue culture vessel was returned to a humidified incubator set to 37°C and 5% CO2. The remainder of the Phase 1 Expansion, Phase 2 Expansion and Monocyte Differentiation, and Phase 3 Macrophage Differentiation cultures proceeded as described in Example 1. Cells were collected and cryopreserved at the end of Phase 2 and the end of Phase 3 of culture for subsequent in vitro analyses.
  • Monocytes and macrophages generated by ex vivo expansion and differentiation of CD 19 CAR-engineered HSPCs express cell surface CD 19 CAR following cryopreservation and thaw of the cells (see FIGS 8A - 8D).
  • the CD19 CAR+ CD1 lb + monocyte population makes up 38 % of the total cell population (see FIGS. 8A and 8B).
  • the CD 19 CAR+ CDl lb + macrophage population makes up 34.8 % of the total cell population (see FIGS. 8C and 8D).
  • the cell engineering process and expression of the CD 19 CAR by the cells do not reduce the total fold expansion (see FIG 9 A) or viability (see FIG 9B) of the cells compared with control untransduced cells.
  • Cryopreserved and thawed monocytes and macrophages expressing a cell surface CD 19 CAR retain their capacity for phagocytosis at similar or improved levels compared with control untransduced monocytes and macrophages (see FIG. 9C).
  • the CD34+ cell source can comprise single cord blood units from non-HLA matched (unmatched), matched, or partially mismatched donors.
  • the CD34+ cell source can comprise pooled cord blood units from unmatched, matched, or partially mismatched donors.
  • the number of pooled donor cord blood units can comprise between 2 and 20, or more.
  • Enriched CD34+ cells can be used fresh, immediately following enrichment, or can comprise cryopreserved and stored in liquid nitrogen vapor phase for use at a later date.
  • the duration of the Phase 1 HSPC Expansion culture can comprise between 3 and 21 days, such as for example, 3 days, 7 days, 10 days, 14 days, or 21 days.
  • the duration of the Phase 2 Expansion and Monocyte Differentiation culture can comprise between 7 and 14 days, such as, for example, 7 days, 10 days, or 14 days.
  • the duration of the Phase 3 Macrophage Differentiation culture can comprise 6 to 8 days.
  • the preferred culture condition comprises a 14-to-21-day Phase 1 culture duration, a 7-day Phase 2 culture duration, and a 7-day Phase 3 culture duration.
  • Base medium used during Phase 1 HSPC Expansion culture can comprise StemSpanTM SFEM II, StemProTM-34 SFM, X- VIVOTM- 10, X- VIVOTM- 15, PRIME-XV Expansion XSFM, CellGenix® SCGM, StemLine® or StemLine® II Hematopoietic Stem Cell Expansion Medium, or StemMACS HSC Expansion Media.
  • Base medium used during Phase 2 Expansion and Monocyte Differentiation culture can comprise StemSpanTM SFEM II, StemProTM-34 SFM, X-VIVOTM-10, X-VIVOTM-15, or IMDM.
  • Base medium used during Phase 3 Macrophage Differentiation culture can comprise X-VIVOTM 10, X-VIVOTM 15, RPMI 1640, IMDM, or ImmunoCultTM- SF Macrophage Medium.
  • the preferred culture condition comprises use of X-VIVOTM- 10 during Phase 1 of culture, X-VIVOTM 10 during Phase 2 of culture, and RPMI 1640 during Phase 3 of culture.
  • Culture media supplements can comprise serum or a serum replacement and/or one or more cytokines.
  • Cytokine supplements used during Phase 1 HSPC Expansion culture comprise 50 ng/mL stem cell factor (SCF), 50 ng/mL thrombopoietin (TPO), 50 ng/mL FMS-like tyrosine kinase 3 ligand (Flt3-E), 50 ng/mE interleukin 6 (IE-6), and 10 ng/mL interleukin 3 (IL-3).
  • SCF stem cell factor
  • TPO thrombopoietin
  • Flt3-E FMS-like tyrosine kinase 3 ligand
  • IE-6 interleukin 6
  • IL-3 interleukin 3
  • Cytokine supplements used during Phase 2 Expansion and Monocyte Differentiation culture can comprise a combination of 50 ng/mL M-CSF, 50 ng/mL Flt3-L, and 10 ng/mL interleukin 3 (IL-3).
  • Cytokine supplements used during Phase 2 Expansion and Monocyte Differentiation may alternately comprise a combination of 50 ng/mL M-CSF, 50 ng/mL SCF, 50 ng/mL TPO, 50 ng/mL Flt3-L, 50 ng/mL IL-6, and 10 ng/mL IL-3.
  • Cytokine supplements used during Phase 2 Expansion and Monocyte Differentiation can alternately comprise a combination of 50 ng/mL M-CSF, 50 ng/mL Flt3-L, 10 ng/mL IL-3, and 50 ng/mL IL-6; it can alternately comprise a combination of 50 ng/mL GM-CSF, 50 ng/mL MCSF, 50 ng/mL Flt3-L, and 10 ng/mL IL-3.
  • Serum or serum replacements used during Phase 3 Macrophage Differentiation culture can comprise, for example, 10 % fetal bovine serum (FBS); 10 % human AB serum; 2.5 % human platelet lysate (HPL), 5 % HPL, or 10 % HPL; or 0.25 % human serum albumin (HSA), 0.5 % HSA, 1 % HSA, or 2 % HSA.
  • FBS fetal bovine serum
  • HPL human platelet lysate
  • HSA human serum albumin
  • Cytokine supplements used during Phase 3 Macrophage Differentiation culture can also comprise, for example, 10 ng/mL GM-CSF, 25 ng/mL GM-CSF, or 50 ng/mL GM-CSF; or 25 ng/mL M-CSF, 50 ng/mL M-CSF, or 100 ng/mL M-CSF.
  • the preferred culture conditions comprise X-VIVOTM- 10 medium supplemented with 50 ng/mL stem cell factor (SCF), 50 ng/mL thrombopoietin (TPO), 50 ng/mL FMS-like tyrosine kinase 3 ligand (Flt3-L), 50 ng/mL interleukin 6 (IL-6), and 10 ng/mL interleukin 3 (IL-3) for Phase 1 HSPC Expansion; X-VIVOTM-10 medium supplemented with 50 ng/mL MCSF, 50 ng/mL Flt3-L, and 10 ng/mL IL-3 for Phase 2 Expansion and Monocyte Differentiation; and RPMI 1640 medium supplemented with 10 ng/mL GM-CSF and either 2.5 % HPL or 10 % FBS for Phase 3 Macrophage Differentiation.
  • SCF stem cell factor
  • TPO thrombopoietin
  • Flt3-L
  • In vitro culture of differentiating cells with feeder cell layers or with a tissue culture vessel substrate or coating may be done to activate cell signaling pathways that influence cell differentiation toward a specific desired lineage or activation status.
  • Phase 1 HSPC Expansion culture cells have been cultured in the presence of no coating or DXI and RetroNectin coating: o for all 7 days of a 7-day Phase 1 culture period; o for the first 7 days of a 14- or 21 -day Phase 1 culture period; and o for the first 14 days of a 21-day Phase 1 culture period.
  • the preferred culture condition comprises tissue culture treated plastic culture vessels with DXI coating for 7 days, 10 days, or 14 days, followed by 7 days without DXI coating for Phase 1 of culture, and using tissue culture treated plastic culture vessels without coating or pre-treatment for Phase 2 and Phase 3 of culture; an alternate culture condition comprises tissue culture treated plastic culture vessels without additional coating or pre-treatment during Phase 1, Phase 2, and Phase 3 of culture.
  • the cells generated through the above are intended for use either as a fresh cell product that has not been cryopreserved or as a cryopreserved cell product.
  • Cells can be cryopreserved following the completion of the first phase of culture, at which point expanded stem and progenitor cells have been generated.
  • a cryopreserved expanded cell product can differentiate to monocytes and macrophages upon thaw and culture under the differentiation culture conditions described herein.
  • the cell product can be used as a fresh monocyte cell product or as a cryopreserved monocyte cell product following the completion of the second phase of culture, at which point differentiated monocytes have been generated.
  • a cryopreserved monocyte cell product can differentiate to functional macrophages and dendritic cells upon thaw.
  • the cell product can be used as a fresh macrophage cell product or as a cryopreserved macrophage cell product following the completion of the third phase of culture, at which point differentiated macrophages have been generated.
  • Both monocytes and macrophages generated through the above can be cryopreserved and thawed with recovery of about 46 to about 85 % of the monocytes with about 66 to about 90 % viability and recovery of about 53 to about 99 % of the macrophages with about 83 to about 99 % viability.
  • Monocytes and macrophages can be cryopreserved using CryoStor® CS 10, and in the alternative the cells can also be effectively cryopreserved using similar cryopreservation media including, but not limited to, CryoStor® CS5, FBS + 5 % DMSO, FBS + 10 % DMSO, human AB serum + 5 % DMSO, human AB serum + 10 % DMSO, HPL + 5 % DMSO, HPL + 10 % DMSO.

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Abstract

La présente invention concerne une méthode ex vivo de production d'une composition ou d'un produit thérapeutiques de monocytes et/ou de macrophages (monocytes/macrophages) destinés à être utilisé en immunothérapie. La méthode comprend trois phases consistant en une phase de multiplication des cellules souches hématopoïétiques et des cellules souches et progénitrices hématopoïétiques (HSPC), une phase de multiplication et de différenciation des monocytes et une phase de différenciation des macrophages. La composition ou le produit de monocytes et/ou de macrophages peuvent être utilisés directement dans la formulation d'un produit thérapeutique destiné à être administré à un sujet ou cryoconservé pour une utilisation et/ou une formulation ultérieures. Le produit de cellules monocytes et/ou macrophages, la composition et/ou le produit peuvent également être ingéniérisés pour comprendre un récepteur (tel qu'un CAR, un TCR, un récepteur chimérique ou un autre récepteur), un recruteur de cellules immunitaires, un anticorps, un nanocorps, un facteur de transcription, une cytokine et/ou une chimiokine. La fonction du produit cellulaire peut également être améliorée par précharge des cellules avec un virus oncolytique ou une autre molécule immunomodulatrice en vue d'une administration à une tumeur solide.
PCT/US2023/073151 2022-09-07 2023-08-30 Méthodes de génération d'un produit de cellules monocytes/macrophages à partir de cellules souches et progénitrices hématopoïétiques cd34+ issues du sang de cordon multipliées WO2024054769A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180010096A1 (en) * 2015-01-16 2018-01-11 Agency For Science, Technology And Research Differentiation of macrophages from pluripotent stem cells
WO2021108769A1 (fr) * 2019-11-27 2021-06-03 Deverra Therapeutics Inc. Compositions et procédés de culture de cellules souches et progénitrices hématopoïétiques

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180010096A1 (en) * 2015-01-16 2018-01-11 Agency For Science, Technology And Research Differentiation of macrophages from pluripotent stem cells
WO2021108769A1 (fr) * 2019-11-27 2021-06-03 Deverra Therapeutics Inc. Compositions et procédés de culture de cellules souches et progénitrices hématopoïétiques

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